Objective:To produce a monoclonal antibody (mAb) against human trophoblast cell-surface antigen 2(Trop2) with hybridoma technology and identify its immune specificity. Methods:The spleen cells from BALB/c mice immunized with pancreatic cancer cell line BxPC3 cells were fused with SP2/0 cells to produce a monoclonal antibody against human Trop2. Further, several immunological methods such as ELISA,IF,IP,MALDI-TOF MS,FACS and IHC were used to characterize the immune specificity of the mAb. Results:The results demonstrated that the specific mouse anti-human Trop2 mAb was successfully produced and can be secreted continuously and steadily. Moreover, this mAb can effectively recognize the transmembrane glycoprotein Trop2 over-expressed in human cancer cells and tissues. Conclusion:We successfully obtained mouse anti-human Trop2 mAb which may be potentially utilized for some tumor targeted therapy.