In order to construct and express human cardiac troponin C-linker-troponin I(P) fusion protein, detect its activity and prepare lyophilized protein, we searched the CDs of human cTnC and cTnI from GenBank, synthesized cTnC and cTnI(30-110aa) into cloning vector by a short DNA sequence coding for 15 neutral amino acid residues. pColdⅠ-cTnC-linker-TnI(P) was constructed and transformed into E. coli BL21(DE3). Then, cTnC-linker-TnI(P) fusion protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG). Soluable expression of cTnC-linker-TnI(P) in prokaryotic system was successfully obtained. The fusion protein was purified by Ni2+ Sepharose 6 Fast Flow affinity chromatography with over 95% purity and prepared into lyophilized protein. The activity of purified cTnC-linker-TnI(P) and its lyophilized protein were detected by Wondfo FinecareTM cTnI Test. Lyophilized protein of cTnC-linker-TnI(P) was stable for 10 or more days at 37℃and 4 or more months at 25℃and 4℃. The expression system established in this research is feasible and efficient. Lyophilized protein is stable enough to be provided as biological raw materials for further research.