In the year 1600, Albani moved to Rome to work in the fresco decoration of the gallery of the Palazzo Farnese, being completed by the studio of Annibale Carracci. Rome, under Clement VIII Aldobrandini (1592-1605) was exhibiting some degree of administrative stability and renewed artistic patronage. While pope Clement was born from a Florentine family residing in Urbino, his family was allied by marriage to the Emilia-Romagna and the Farnese, since Ranuccio I Farnese, Duke of Parma had married Margherita Aldobrandini. Parma, like Bologna, are part of the Region of Emilia-Romagna. Thus it was not a surprise that Cardinal Odoardo Farnese, Ranuccio's brother, chose the Carraccis from Bologna for patronage, thereby establishing Bolognese predominance of Roman fresco painting for nearly two decades. Albani became one of Annibale's most prominent apprentices. Using Annibale's designs and assisted by Lanfranco and Sisto Badalocchio, Albani completed frescoes for the San Diego Chapel in San Giacomo degli Spagnoli between 1602-1607. In 1606-7, Albani completed the frescoes in the Palazzo Mattei di Giove in Rome. He later completed two other frescoes in the same palace, also on the theme of Life of Joseph.

In 1609, he completed the ceiling of a large hall with Fall of Phaeton and Council of the Gods for the Palazzo Giustiniani (now Palazzo Odescalchi) at Bassano (di Sutri) Romano. This work was commissioned by the Marchese Vicenzo Giustiniani, famous for also being patron to Caravaggio. During 1612-14, Albani completed the Choir frescoes at the newly remodeled (by Pietro da Cortona) church of Santa Maria della Pace. In 1616 he painted ceiling frescoes of Apollo and the Seasons at Palazzo Verospi in Via del Corso for the cardinal Fabrizio Verospi.

In later years, Albani developed a mutual, though respectful, rivalry with the more successful Guido Reni, who was also heavily patronized by the Aldobrandini, and under whom Albani had worked at the chapel of the Palazzo del Quirinale.

Albani's best fresco masterpieces are those on mythological subjects. Among the best of his sacred subjects are a St Sebastian and an Assumption of the Virgin, both in the church of San Sebastiano fuori le Mura in Rome. He was among the Italian painters to devote himself to painting cabinet pictures. His mythological subjects include The Sleeping Venus, Diana in the Bath, Danaë Reclining, Galatea on the Sea, and Europa on the Bull. A rare etching, the Death of Dido, is attributed to him. Carlo Cignani, Andrea Sacchi, Francesco Mola, and Giovanni Francesco Grimaldi were some of his students. On the death of his wife he returned to Bologna, where he married a second time and resided till his death.

Albani never acquired the monumentality or tenebrism that was quaking the contemporary world of painters, and in fact, is derided often for his lyric, cherubim-filled sweetness, which often has not yet shaken the mannerist elegance. While Albani's thematic would have appealed to Poussin, he lacked the Frenchman's muscular drama. His style sometimes appears to befit the decorative Rococo more than of his time. Among the pupils of Albani were his brother Giovanni Battista Albani, and others including Giacinto Bellini, Girolamo Bonini, Giacinto Campagna, Antonio Catalani, Carlo Cignani, Giovanni Maria Galli, Filippo Menzani, Andrea Sacchi, Andrea Sghizzi, Giovanni Battista Speranza, Antonio Maria del Sole, Emilio Taruffi, and Francesco Vaccaro.