Abstract: Objective To express the human recombinant SUMO1 protein and prepare monoclonal antibody (mAb) against it. Methods The recombinant expression plasmid pET32a-HIS-SUMO1 was made and transformed into E.coli (BL21), and then the recombinant fusion protein HIS-SUMO1 was expressed and purified. The BALB/c mice were immuned with pure protein HIS-SUMO1 as antigen. Monoclonal antibody against SUMO1 were prepared by using standard hybridoma technology. The hybridoma cell lines were obtained by ELISA and Western Blot screening procedure, the isotype of the mAbs were further identified by immune-double diffusion. Ascites were prepared from one propagated hybridoma cell line and mAbs were purified by using the Kit of Millipore. The valence of mAb was detected by Western Blot. Results The recombinant protein HIS-SUMO1 is expressed and purified . Three hybfidmas producing antibodies against SUMO1 were obtained, the isotypes of three mAbs are IgG1, Western Blot showed that the antibodies were specific for SUMO1. The antibody purified from the ascites has better specificity. Conclusion The SUMO1 mAb prepared by using recombinant SUMO1 protein as antigen can be used for detecting the protein sumoylation.