Loss or mutation of antigen and limited chimeric antigen receptor (CAR) T-cell persistence in vivo are essential determinants of recurrence in relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL). Sequential infusion of CD19 and CD20 CAR Tcells (sequential CD19-20 CAR T-cell therapy) have been proposed as a potential solution. From March 2019 to January 2022 in Beijing Gobroad Boren Hospital, a total of 21 patients with r/r DLBCL received the CD19-20 CAR T-cell therapy in the prospective study (Clinical Trials Number: ChiCTR1900020980).Grade ≥3 cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) were observed in 9.5% (2/21) and 0% (0/21) of patients, respectively. The CD20 CAR T-cell infusion did not increase severe toxicity. There were no treatment-related deaths. Of 21 patients, 13 (61.9%) attained partial responses (PRs) and 8 (38.1%) attained complete responses (CRs) within 90 days after CD19 CAR T-cell infusion. Subsequent treatment with CD20 CAR T-cell infusion resulted in 10 of the 13 initial PR patients converting to CR, with a median time to achieving CR of 30 days (range, 30-90). With a median follow-up of 24.7 months (range, 11.6-45.86), 71.4% (15/21) of patients maintained ongoing responses at the data cutoff date. Overall, sequential CD19-CD20 CAR T-cell therapy demonstrated a favorable safety profile and might enhance long-term clinical outcomes.