Objective To express cardiac troponin I(cTnI) and cTnC in E. coli system,prepare cTnI-cTnC complex antigen and validate its performance. Methods The synthetic cTnI and cTnC genes were inserted into prokaryotic expression vectors pET-22b and pET-28a resp.,and the constructed recombinant plasmids were transformed to E. coli BL21(DE3) for induction with IPTG. The expressed cTnI and cTnC proteins were purified by nickel ion affinity chromatog. and ion-exchange chromatog. and mixed at a mole ratio of 1 : 1 in presence of calcium ion. The antigenic reactivity of the prepared cTnI-cTnC complex protein was determined by double antibody sandwich ELISA. The cTnI and cTNI-CTnC complex protein were diluted and stored at 4 and 37 °C for 14 d sep.,of which the concentrations before and after storage were determined to validate the stability. Results Both the SDS-PAGE purity of purified cTnI and cTnC were more than 90%. Compared with control antigen(8IC63 complex antigen),CTnI-cTnC complex protein showed strong reaction signal with the antibody,and was recognized by cTnI and cTnC antibodies simultaneously. In accelerated stability test at 37 °C,the concentration of cTnI protein decreased by about 80%,while the concentration change of cTnI-cTnC complex protein was ± 15%. Conclusion Prepared cTnI-cTnC complex protein showed good antigen reactivity and stability,which provided a reliable material for preparation of highly reactive and stable calibrators of cTnI assay kit.