After serving in the Italian military, Di Stefano made his operatic debut in 1946 in Reggio Emilia as Des Grieux in Massenet's Manon, the role in which he made his La Scala debut the following year. The great beauty of his lyric tenor voice quickly won him international attention and he was duly engaged by the the Metropolitan Opera in New York. He made his New York debut in 1948 as the Duke in Rigoletto. He went on to perform regularly in New York for many years. In 1957, Di Stefano made his British debut at the Edinburgh Festival as Nemorino in L'elisir d'amore and his Royal Opera House, Covent Garden, debut in 1961, as Cavaradossi in Tosca.
As a singer, Di Stefano was admired for his excellent diction, unique timbre, passionate delivery and, in particular, for the sweetness of his soft singing. In his Metropolitan Opera radio-broadcast debut in Faust, he attacked the high C forte and then softened to a pianissimo. Sir Rudolf Bing said in his memoirs that this was the most beautiful sound he had heard come out of a human throat during his many years as general manager of the Metropolitan Opera.
But the heavier roles that Di Stefano began to take on during the 1950s were not really suitable for a lyric tenor. His voice deteriorated and, by the mid-1960s, he had all but ended his operatic career.
During his years of international celebrity, he won a gold Orfeo, an Italian musical award similar to America's Oscar.
In 1973, Di Stefano accompanied Callas on her final recital tour, an undertaking that was eventually aborted in 1974 due to the vocal shortcomings of both singers. His final operatic role was as the aged Emperor in Turandot, in July 1992.
Tenor Luciano Pavarotti said he modelled himself after Di Stefano, a fact that gained much attention after Pavarotti’s death in September 2007. However, on the 1992 PBS television program "Pavarotti and the Italian Tenor," a vocal coach explained that Di Stefano's technique was fundamentally faulty because he did not use the passaggio in his voice as he should have. Nonethless, Di Stefano still managed to enjoy a significant operatic career, despite facing stiff competition during the 1950s and '60s from such outstanding fellow tenors as Mario Del Monaco, Franco Corelli, Carlo Bergonzi, Nicolai Gedda, Jon Vickers, Richard Tucker and, prior to 1960, Jussi Bjorling.
A series of duets with Giuseppe di Stefano and Maria Callas were recorded by the Philips label in the period November-December 1973 with Antonio de Almeida conducting the London Symphony Orchestra. These recordings were not published officially but a 'pirate' version did appear.
For English DECCA he recorded L'elisir d'amore with Hilde Güden and Fernando Corena (1955), La Gioconda (with Zinka Milanov and Leonard Warren, 1957), La forza del destino (1958) and a second Tosca (with Leontyne Price and Giuseppe Taddei, von Karajan conducting, 1962).
For Ricordi (Ricordi MRO 104/105), he made a complete stereo Lucia di Lammermoor with Renata Scotto, Ettore Bastianini and Ivo Vinco in 1958, with Nino Sanzogno conducting the orchestra and chorus of the Teatro alla Scala, Milan.
In 1995, VAI issued an approved version of La bohème, from a 1959 performance in New Orleans, with the tenor starring opposite Licia Albanese, Audrey Schuh, Giuseppe Valdengo and Norman Treigle. Additionally, in 1962 the tenor recorded excerpts from Massenet's Manon, with Anna Moffo, conducted by René Leibowitz.
In 1951, Di Stefano sang in a performance of the Verdi Requiem, at Carnegie Hall, conducted by Arturo Toscanini, other soloists being Herva Nelli, Fedora Barbieri and Cesare Siepi. It was released by RCA.