Of a noble family (high grand-uncle was a cardinal), he entered the service of prince François, Duke of Anjou, the younger brother and rival of Henri III. Bussy became well-known at court as Anjou's "favorite" and later became the lover of princess Marguerite de Valois.

Bussy d'Amboise once went to the Louvre Palace dressed very plainly with twelve valets in gold liveries. When asked why he was dressed so poorly in comparison with his liveries, he responded "When valets dress like gentlemen, the only thing left to do for gentlemen is to dress like valets", a comment directed towards the rest of the favourites who were dressed in their finest garments.

Bussy caused another stir during the parties organised by King Henri III for the wedding of one of his favourites, Saint Luc, and Bussy was forced into a temporary exile. He was the lover of king Henry III's brother the duc François d'Alençon.