With the self-developed two-photon fluorescent probe glycyl-prolyl-N-butyl-4-amino-1,8-naphthalimide(GP-BAN) for dipeptidyl peptidase-Ⅳ(DPP-Ⅳ) detection with high specificity and sensitivity as the substrate and human plasma as the source of DPP-Ⅳ，this study aims to evaluate the inhibitory effect of Qingfei Paidu Decoction and the medicinals in the prescription on DPP-Ⅳ，and to further explore the effective compounds in the medicinals.Methods:With the established DPP-Ⅳ inhibitor screening system，the solution of Qingfei Paidu Decoction powder，the aqueous solution of the Chinese medicinal granules，and the effective compounds in the medicinals were respectively incubated with GP-BAN at 37 ℃.The inhibition on DPP-Ⅳ was evaluated based on the fluorescence value of the probe metabolite BAN.Results:Qingfei Paidu Decoction showed mild inhibitory effect on DPP-Ⅳ.The aqueous solution of Herba Ephedrae，Fructus Aurantii Immaturus，and Herba Pogostemonis granules at 1 000 μg/mL had obvious inhibitory effect on DPP-Ⅳ，and the residual activity of the enzyme was 40.00%，61.05%，and 62.04%，separately.Hesperetin，rutin，apigenin，quercetin，kaempferol，norephedrine，and nobiletin significantly suppressed DPP-Ⅳ，particularly quercetin，kaempferol，and apigenin whose half maximal inhibitory concentration(IC50) on DPP-Ⅳ was(75.39±6.17) μmol/L,(9.16±0.69) μmol/L，and(8.29±0.93) μmol/L，respectively.Conclusion:Qingfei Paidu Decoction inhibits DPP-Ⅳ，and the medicinals in the prescription and the compounds in the medicinals also show inhibitory effect on DPP-Ⅳ.These findings can serve as a reference for the treatment of coronavirus disease 2019(COVID-19) with Qingfei Paidu Decoction，especially COVID-19 complicated with type 2 diabetes mellitus(T2DM).