CD19‐targeted chimeric antigen receptor T (CAR‐T) cells using murine single‐chain variable fragment (scFv) has shown substantial clinical efficacy in treating relapsed/refractory acute lymphoblastic leukemia (R/R ALL). However, potential immunogenicity of the murine scFv domain may limit the persistence of CAR‐T cells. In this study, we treated 52 consecutive subjects with R/R ALL with humanized CD19‐specific CAR‐T cells (hCART19s). Forty‐six subjects achieved complete remission (CR) (N = 43) or CR with incomplete count recovery (CRi) (N = 3) within 1 month post infusion. During the follow‐up with a median time of 20 months, the 1‐year cumulative incidence of relapse was 25% (95% confidence interval [CI] 13–46), and 1‐year event‐free survival was 45% (95% CI 29–60). To the cutoff date, 20 patients presented CD19+ relapse and 2 had CD19− relapse. Among the 22 relapsed patients, 14 had treatment‐mediated and treatment‐boosted antidrug antibodies (ADA) as detected in a sensitive and specific cell‐based assay. ADA positivity was correlated with the disease relapse risk. ADA‐positive patients had a significantly lower CAR copy number than ADA‐negative patients at the time of recurrence (p < .001). In conclusion, hCART19s therapy is safe and highly active in R/R ALL patients, and the hCART19s treatment could induce the emergence of ADA, which is related to the recurrence of the primary disease.