He appears to have learned his trade from painter Antonio de Lanchares, who died in 1630. He then spent time in El Escorial, where his particular attention to the Venetian canvases allowed him to acquire a technique so close to that of Titian that some of his works—“Saint Stephen” (Musée des Beaux-Arts de Lille), for example— have been mistaken for those of that master from Cadore. He is known to have also received commissions for the Alcázar in Madrid, and he made a pair of canvases, now lost, of former monarchs for the palace's Golden Hall. The Museo del Prado has three paintings linked to Diego Polo: “The Penitent Saint Jerome”, which belonged to the infante don Sebastián Gabriel de Borbón's collection and was acquired by the Prado in 1982; “Saint Roch”, which was purchased as an anonymous work in 1965 and later attributed to Diego Polo by Alfonso E. Pérez Sánchez; and his best-known work, “The Gathering of Manna”, which he painted for the scribe of Madrid, Alonso Portero. Period sources quote praise of this work by no less than Velázquez himself, and it is undoubtedly the most explicit example of Polo's knowledge of Venetian color. A drawing for that composition is now at the Galleria degli Uffizi in Florence (García López, D. in Enciclopedia M.N.P, 2006, vol. V, p. 1774).