Objective To prepare monoclonal antibodies against human cardiac troponin I (cTnI). Methods Balb/c mice were immunized by human cTnI as the antigen, and the high affinity and specificity monoclonal antibody against human cardiac troponin I was prepared through hybridoma technology. The titer of the antiserum, purified by protein G affinity of antibody, was then determined by indirect ELISA. The antigen binding site and the affinity were analyzed by competitive ELISA. SDS-PAGE and Western blotting were used to identify the specificity. Results Total 9 strains of hybridoma cell lines secreting monoclonal antibody against cTnI were obtained, and the antibody secreted by A3, A9 were of high purity and its immunoglobulin subclasses was IgG2a. There was no cross reaction with CK-MB, cTnT. Both titers were 1:1024000, and the affinity were 4.21 x 108 mol/L and 1.07 x 108 mol/L respectively. The antigen binding sites for them were different. Conclusion A pair of high affinity and specificity of monoclonal antibody against human cardiac troponin I was prepared successfully.