Objective To prepare and characteriz the monoclonal antibody against Mb to provide basis for the development of Mb diagnostic reagent. Methods BALB/c mice were immunized with human myoglobin (Mb) antigen, the positive hybridoma were obtained by using hybridoma cell technique. The characterization of antibody were determined by ELISA. Results Nine monoclonal antibodies against Mb were prepared. The titers of ascites were 1:5×104～1:2×105. Five monoclonal antibodies successfully formed six anatibody pairs. The affinity constants of five monoclonal antibodies were in the range of 6.46×108 to 3.68×109 L/mol. The cross-reaction ratios with HB and ALB were 7.434×10-3%～2.904×10-2% and 4.980×10-7%～3.830×10-5% respectively. Two monoclonal antibodies (Mb17B6 and Mb21E8) were used to develop the chemiluminescence immunoassay. The assay sensitivity was 6.02 ng/mL. Conclusion The monoclonal antibodies against Mb are prepared successfully for the development of immunoassay.