Fully human antibody fragment Fab that specifically binding to Trop-2 (trophoblast cell-surface antigens 2, Trop-2), was selected from phage display antibody library. Positive phage-displayed antibody clones were selected on live cell lines and immobilized protein. The purified Fab was verified by SDS-PAGE and Western blot, which showed two bands at about 28 ku and 32 ku at the expected sizes. To analyze the immunological characters of Fab for Trop-2 binding, flow cytometry, immunoprecipitation assays and mass spectrometry were set up and carried out with BxPc3 and NIH3T3 cell lines. The results demonstrated Fab could bind native Trop-2 specifically on the BxPc3 cell surface. Peptide mapping fingerprint showed that the protein which bound is Trop-2 protein. Immunohistochemistry detection illuminated Fab could bind the membrance protein of pancreatic cancer tissue. In vitro cell growth inhibition assay showed that anti-Trop-2 Fab could inhibit the Trop-2 positive cell BxPc3 growth, it illuminated that anti-Trop-2 Fab bound the Trop-2 on Trop-2 positive cell surface. For Trop-2 negative cell line NIH3T3, no significant inhibition among the different dosages of Fab. The results showed that anti-Trop-2 Fab antibody fragments could recognize Trop-2 extracellular domain in native conformation, making them as potential powerful reagents for clinical therapeutic application.