Abstract: Dipeptidyl peptidase 4 (DPP4) is a serine protease widely present in human body, which can specifically catalyze the hydrolysis of proline (Pro) or alanine (Ala) peptide bond at the second N-terminal position of polypeptide chain. It is involved in the activation, partial or complete inactivation of a variety of bioactive polypeptides in vivo. In this study, the kinetic parameters of different probe substrates GP-PNA, GP-AMC and GP-BAN catalyzation by DPP4 were compared, and the inhibitory kinetic differences between DPP4 and its inhibitor sitagliptin/vildagliptin under different probe systems were studied. By means of molecular docking technology, the interaction mechanism between DPP4 and different substrates was revealed from the perspective of small molecules, and the key factors affecting the activity of DPP4 catalytic substrates were discussed. The results show that DPP4 has the best binding property to probe GP-BAN, and its affinity is higher than that of GP-AMC and GP-PNA, which proves that the carbon structure of probe is the key factor affecting the binding. Sitagliptin has better inhibitory activity on DPP4 than vildagliptin. The molecular docking results suggest that the halogenation characteristic of sitagliptin may be the key factor to enhance its binding ability. This study can provide an important basis for the selection of DPP4 probes under different research purposes, and also provide experimental data and research basis for rational design and development of highly selective DPP4 ligand tool molecules and personalized rational drug use in clinic.