After studying at a German-language grammar school in Žatec (Saaz), he was admitted at Prague University where he obtained a degree as a doctor of medicine (1872).

After eight months, Holub set out in a convoy of local hunters on a two-month experimental expedition, or "scientific safari", where he began to assemble a large natural history collection.

In 1883, Holub, along with his new wife Rosa (1865–1958) and six European guides, set out to do what no one had done before: explore the entire length of Africa from Cape Town all the way to Egypt.

However, the expedition was troubled by illness, poisoning of draugh oxen by plant Dichapetalum cymosum and, eventually, the uncooperative or even rebelling Ila tribesmen and Holub's team was forced to turn back in 1886.

Frustrated that he was unable to find a permanent home for his large collection of artefacts, he gradually sold or gave away parts of it to museums, scientific institutions and schools.