ABSTRACT Objective Taking Lonicera japonica Thunb. as the research object, it has the effect of clearing away heat and detoxifying, the anti-RNA virus protease activity of L. japonica Thunb.was analyzed based on molecular docking and experimental verification. Methods Construction of small molecule ligand library of L. japonica Thunb. by TCMSP (Traditional Chinese Medicine Systems).The binding of L. japonica to HIV-1 protease and Cathepsin L protease was analyzed using drug molecular design simulation software SYBYL 2.1.1. The activities of different solvent extracts (water extract, 30% alcohol extract, 60% alcohol extract and 85% alcohol extract) of L. japonica were verified by fluorescence method against HIV-1 and Cathepsin L protease.Results Twenty-one active components were obtained from L. japonica Thunb. using TCMSP, and 20 of them had good binding with HIV-1 protease,the anti-HIV-1 protease activity of L. japonica Thunb. was stronger than Cathepsin L protease by molecular docking. By fluorescence analysis, it was found that the water extract, 30% alcohol extract, 60% alcohol extract and 85% alcohol extract had anti-HIV-1 activity with IC50 values of 0.14 mg/mL, 0.015 mg/mL. 0.05 mg/mL and 0.12 mg/mL. All extracts no inhibition against Cathepsin L protease. The experimental results are in good agreement with computer screening results.Conclusions This study preliminarily confirmed the activities of anti-HIV-1 protease and Cathepsin L protease in L. japonica Thunb., and the results provide theoretical basis for the development and application of antiviral drugs.