Abstract: The aims are to express starch phosphorylase in vitro, and prepare monoclonal antibodies in immunized mice. Starch phosphorylase genes of cassava were amplified by polymerase chain reaction (PCR) and then cloned into the prokaryotic expression vector (pET28a), the starch phosphorylase was expressed in E. coli and then purified. The purified starch phosphorylase was used to immunize Babl/c mice and the titer of mice serum was determined by indirect ELISA. Mouse spleen cells and murine SP2/0 cells were fused, and then hybridoma cell lines which could produce starch phosphorylase monoclonal antibody were prepared, the subtypes and stability of antibody were detected. Recombinant plasmid in E.coli could express starch phosphorylase efficiently, SP2/0 cells and spleen cells of 3# mice were fused, then 15 strains with antibody titer more than 105 were obtained, and the antibodies could against starch phosphorylase stably, and there was no cross reaction with calmodulin and bovine serum albumin, in which 13 strains were IgG type, and 2 strains were very stable. The monoclonal antibody of starch phosphorylase had high titer, strong specificity, and good stability, it could provide a basis for starch phosphorylase study on cassava.