Impressive outcomes have been achieved by chimeric antigen receptor (CAR)‐T cell therapy using murine‐derived single‐chain variable fragment (scFv) FMC63 specific for CD19 in patients with B cell malignancies. However, evidence suggests that human anti‐mouse immune responses might be responsible for poor persistence and dysfunction of CAR‐T cells, leading to poor outcomes or early tumor recurrence. Substituting a fully human scFv for murine‐derived scFv may address this clinically relevant concern. In this study, we discovered two human anti‐CD19 scFv candidates through an optimized protein/cell alternative panning strategy and evaluated their function in CAR‐T cells and CD19/CD3 bispecific antibody formats. The two clones exhibited excellent cytotoxicity in CAR‐T cells and bispecific antibodies in vitro compared with the benchmarks FMC63 CAR‐T cells and blinatumomab. Furthermore, Clone 78‐BBz CAR‐T cells exhibited similar in vivo antitumor activity to FMC63‐BBz CAR‐T cells. Our results indicate that Clone 78‐BBz CAR has excellent efficacy and safety profile and is a good candidate for clinical development.