He was trained in the academy of Lodovico Carracci (Carracci, Lodovico) and in 1602 was in Rome, where he joined the Bolognese artists at work under the direction of Annibale Carracci (Carracci, Annibale) in the decoration of the Farnese Palace (Farnese, Palazzo). He was employed by Cardinal Pietro Aldobrandini to decorate a room in the Villa Belvedere at Frascati with mythological frescoes and by Cardinal Farnese to paint a chapel in the Badia at Grottaferrata. Both fresco cycles reflect the spirit of Carracci, though their more rigidly classical schemes look forward to the Scenes from the Life of St. Cecilia that Domenichino painted between 1615 and 1617 for San Luigi dei Francesi and which are among his most successful works. His altarpiece of the Last Communion of Saint Jerome (1614) shows his concern for accurate facial expressions and tightly knit groupings of figures.

In 1617–18 Domenichino painted for Cardinal Aldobrandini the celebrated canvas of The Hunt of Diana, which was subsequently taken by Cardinal Scipione Borghese. This work shows that he was a sensitive colourist, and its idyllic mood departs from the arid classicism of his frescoes. Between 1624 and 1628 he was occupied with the frescoed pendentives and apse of Sant'Andrea della Valle. Though Domenichino's work remained in great demand, there was a reaction against the rigid classicism of his style, and in the last works that he produced in Rome—four frescoed pendentives in San Carlo ai Catinari (1628–30) and the Martyrdom of St. Sebastian for St. Peter's, now in Santa Maria degli Angeli—he worked in a broader, less classical style. This closer approximation to the Baroque is also apparent in the frescoed Scenes from the Life of St. Januarius (1631–41). In Naples his style was less acceptable than it had proved in Rome, and he appears to have been the victim of systematic persecution by local artists, such as José de Ribera (Ribera, José de).

Throughout the 17th and 18th centuries Domenichino's paintings were regarded as second only to those of Raphael, but in the mid-19th century he fell from favour. His importance as a Baroque classicist was recognized again only in the 20th century. His work is marked by lucid and balanced compositions, even and serene lighting and subdued colours, and the sober expressions and restrained gestures of its figures. Domenichino also occupies an important place in the history of landscape painting because his work had a profound influence on the classical landscapists Nicolas Poussin (Poussin, Nicolas) and Claude Lorrain.