Harbour Mice^® HCAb mice were immunized with human Trop2 proteins or overexpression cells. The mice with good anti-Trop2 titer were selected for antibody discovery by single B cell cloning (SBC) platform using Berkeley Lights Beacon^®. 30000+ plasma cells are screened through 3 rounds of SBC, and 529 positive individual cells were exported. After single cell sequencing and CDR analysis, 76 unique HCAbs were selected for gene synthesis and the recombinant HCAbs were transient expressed in 24-well plate with HEK293T cells. Further screening of these recombinant HCAbs were performed using supernatants from 24-well culturing. The binding activities were assessed with Trop2-expressing cell lines, including CHOK1-hTrop2, CHOK1-cynoTrop2, HCC1954, and BxPC-3 by flow cytometry. 21 HCAbs were then selected for 100-ml scale production, and characterization. Finally, 14 HCAb candidates with good binding activity to human Trop2 and cross-reactivity cyno Trop2 were determined.