She was a Victoria's Secret Angel from 2004 to 2017 and has modeled for fashion houses such as Christian Dior, Armani, Ralph Lauren, and Next.

Ambrosio was ranked number five on Forbes' list of the highest-paid models, estimated to have earned $6.6 million every year.

[20] Ambrosio has since appeared in advertising campaigns for Gucci, Dolce & Gabbana, Calvin Klein, Oscar de la Renta, Christian Dior, Escada, Fendi, Giorgio Armani, Guess, Emporio Armani, Moschino, Gap, Hugo Boss, Ralph Lauren, Saks Fifth Avenue, Macy's, Revlon, and the Pirelli Calendar.

[9][21] She has walked the catwalks for designers such as Prada, Chanel, Dolce & Gabbana, Givenchy, Christian Lacroix, Bottega Veneta, Escada, Tommy Hilfiger, Christian Dior, Marc Jacobs, Louis Vuitton, Balmain, Ralph Lauren, Halston, Vivienne Westwood, Giles Deacon, Kenzo, Emanuel Ungaro, Philipp Plein, and Oscar de la Renta.

[29] On 12 August 2012, she appeared in the 2012 Summer Olympics closing ceremony representing the Brazilian fashion tradition on the handover segment to Rio de Janeiro City.

[30] In spring 2014, Ambrosio launched her own fashion and lifestyle brand named Ále by Alessandra[31] in a collaboration with American retailer Cherokee.

[33] For this brand, Ambrosio used her intellectual property company, Silver Sunrise LLC, which works with Cherokee as its global licensing agent.

[34] Ambrosio was chosen by NBC alongside fellow Brazilian model Adriana Lima as the channel's food and culture correspondents for the 2016 Summer Olympics.

The Floral Fantasy Bra, crafted by London Jewellers featured amethysts, rubies, sapphires, and diamonds all set in rose and yellow gold and adorned with more than 5,200 precious stones.

[18][48] Ambrosio guest-starred on the How I Met Your Mother episode "The Yips" on 26 November 2007 with her fellow Victoria's Secret Angels Adriana Lima, Selita Ebanks, Marisa Miller, Miranda Kerr, and Heidi Klum.

[50] Ambrosio made her acting debut in the Brazilian telenovela Verdades Secretas, which premiered on Rede Globo on 8 June 2015, playing the role of former top model Samia.

[57] In March 2019, Ambrosio launched her beachwear brand, GAL Floripa, in partnership with her sister, Aline, and her childhood friend, Gisele Cória.