Objective: To prepare monoclonal antibody (McAb) against the Integrin α6 extracellular domain and identify its biological activities. Methods: Fusion-protein of integrin α6 extracellular domain (GST-IAGED) was expressed in E.coli. JM109 and used for immunizing BALB/C mice. The spleen cells from immunized mice were fused with SP2/0 cells and selectively cultured with HAT medium. ELISA and immunocytochemistry staining were used to select hybridomas. Results: One strain of hybridoma cells that secreted specific monoclonal antibody against integrin α6 extracellular domain was indentified. The immunoglobulin subclass of the McAb was IgG1. Conclusion: The McAb against the extracellular domain of integrin α6 was successfully prepared by using GST-IA6ED fusion protein expressed by E.Coli. And the McAb had positive reaction with human hepatocarcinoma cells-BEL-7402.