The museum was established in 1919 according to the will of art collector Bohdan Khanenko (1917) and the deed of gift to the Ukrainian Academy of Sciences signed by his wife Varvara in 1918.

The art collection of Bohdan and Varvara Khanenko, distinguished Ukrainian collectors and philanthropists of the late 19th and early 20th century, is the core of the museum's holdings.

A group of unique early Byzantine "Sinai" icons created in the 6th and the 7th century has been on display in a separate room of the building since 2004.

The Khanenko Museum's collection includes original artworks by outstanding European masters, such as Pieter Paul Rubens, Gentile Bellini, Juan de Zurbarán, Jacques-Louis David, François Boucher.

Varvara Khanenko was fond of old Italian painting, maiolica, ancient Ukrainian icon, and folk art.

In the late 19th and first decades of the 20th centuries, she was among the Ukrainian cultural figures who started a handicraft movement aimed at giving a new life to folk art traditions.

They travelled extensively throughout Europe, attended auctions, visited private collections, consulted with leading art historians.

In December, 1918, Varvara Khanenko signed the Deed of Gift, by which she handed over the collection, house and library to the Ukrainian Academy of Sciences free of cost.

However, in 1924, two years after Varvara's death, the names of the Khanenkos were removed from the museum's title "due to the lack of revolutionary services to the proletarian culture".

In the early 1920s, the newly opened museum received artworks from other nationalised collections of aristocrats, such as the Repnins, the Branytskis, the Hudym-Levkovyches, the Sakhnovskis.

In 1925, in accordance with the last will of the Saint Petersburg art collector Vasyl Shchavinskyi, his unique collection of Northern European artworks was transferred to the museum.

Among them were a French tapestry featuring the Adoration of the Magi, paintings by European Old Masters, including the diptych "Adam and Eve" (1512) by Lucas Cranach the Elder, a collection of golden items from the Kyivan Rus period, works of Iranian art (the 7th-century silver chalice and the 13th-century aquamanile in the shape of a Zebu cow and a predator).

In 1936, the institution received a collection of Central and Eastern Asian religious artworks; in 1940 — the world-renowned masterpieces: four early Byzantine encaustic icons dating from the 6th and the 7th centuries.

In the summer of 1941, World War II began on the Soviet Union's territory, the most valuable part of the collection was evacuated to the city of Ufa (then the Bashkir ASSR, now Bashkortostan).

In the mid-1990s, the new management led by Vira Vynohradova started the era of restoration of historical memory and intensive development in the history of the museum.

Over the course of the 1990s and the 2000s, the Asian art collection was supplemented with valuable donations from Halyna Scherbak, Vasyl Novytskii, and Oleksandr Feldman.

Today it encompasses the collections of European, Asian and Ancient fine and decorative arts as well as the group of early Byzantine "sinai" icons.

In particular, the museum's collection of paintings includes "Orpheus and Eurydice" by Jacopo da Sellaio, "The God of The Scheldt River, Cybele, and the Goddess of The City of Antwerp" by Peter Paul Rubens, "Still Life with a Chocolate-Grinder" by Juan de Zurbaran, "Burial of A Monk" by Alessandro Magnasco, "Lovers, or Prodigal Son and a Prostitute" by Pierre Louis Goudreaux, "Portrait of Stanislaw August Poniatowski" by Élisabeth Vigée Le Brun, along with paintings by artists from the circles of Hieronymus Bosch, the Bellini brothers, Juan Bautista Martínez del Mazo.

The most notable areas of Asian art collection include Chinese paintings, ceramics (grave figurines of the Tang era, stonewares of the Song period as well as various porcelain items), bronze items, examples of lacquerware and stone carving, Japanese woodcut prints (ukiyo-e), tsuba (artfully decorated traditional sword guards) as well as netsuke miniature sculptures.

The outstanding works of Islamic arts include Iranian lustre-painted pottery and metalwork, miniature paintings, carpets etc.

The historic mansion of Bohdan and Varvara Khanenko located in Kyiv at 15 Tereshchenkivska Street is one of the city's precious architectural landmarks.

Its history goes back to the early 1880s, when the Ukrainian "sugar king" Nykola Tereshchenko, the father of Varvara Khanenko, purchased a large plot of land with a three-storeyed house on a new Kyiv street, which was then called Oleksiivska.

Apart from Robert Meltzer, the following architects and artists contributed to the design of the Khanenkos' mansion: Leonardo Marconi, Pyotr Boitsov, Wilhelm Kotarbinski, Mikhail Vrubel, and Adrian Prakhov.

Judging from archival photos of the house, artworks in the Khanenkos' private art gallery were often rearranged depending on the changes in the couple's interests or their new acquisitions.

In 1919, Varvara Khanenko and the art historian Georgy Lukomsky curated the first public museum exhibition which opened on the ground and the first floors of the mansion.

According to unconfirmed data, during World War II, the Nazi Officers Club was housed in the museum.

Then the mansion was damaged, which made the museum's management dismantle part of the Khanenkos' historic architectural settings.

In addition to this, a covered passage at the first floor level was built to connect the Khanenko Mansion and the Sakhnovski House.

The ground floor houses the Khanenko Museum's scientific library and temporary exhibition rooms as well as a hallway, a ticket office, a wardrobe, and a shop.