Abstract： Objective:To construct an in vitro screening system of Chinese medicine inhibitors of SARS-CoV-2 Mpro by fluorescence resonance en-ergy transfer(FRET)technology.Methods:The engineering plasmids pGEX-4T-1-Mpro and pET-28a(+)-Ls-mK were constructed.The bi-ological activity and enzyme kinetic parameters of Mpro were determined by the fluorescent probe Ls-mK,and the screening conditions were optimized.The differences between fluorescent probe Ls-mK and commercial probe in specificity and detection ability were evaluated by the positive drug GC-376 and the negative control papain-like protease(PLpro)and tobacco etch virus(TEV)protease.According to the con-structed screening system,the inhibitory ability of five Chinese medicine preparations against Mpro was investigated,including Reduning(热毒宁)Injection,Xuebijing(血必净)Injection,Antiviral Oral Liquid and Pudilan(蒲地蓝)Oral Liquid.Results:The engineering plasmid expressed in prokaryotes was successfully constructed,and the recombinant protein fluorescent probes Ls-mK and Mpro with a purity of about 90％were obtained.The fluorescent probe Ls-mK produced FRET phenomenon and presented good specificity.Mpro had good biological ac-tivity.The screening system constructed in this paper specifically had the same detection ability as commercial probes.It was found that only Xiyanping Injection inhibited Mpro,with an IC50 value of 3.32±0.03 mg/mL.Conclusion:This paper successfully constructed a simple,sen-sitive and low-cost screening system based on SARS-CoV-2 Mpro,which laid an experimental foundation for the screening and discovery of SARS-CoV-2 Mpro inhibitors.