Objective To identify novel small molecule inhibitors of SENP1 by virtual screening and evaluate the biological activity in vitro. Methods The possible small molecule inhibitors of SENP1 were selected from the small molecule database by virtual screening. Screened JK043 were chemically synthesized and the inhibitory effect of JK043 on the activity of SENP1 was evaluated by the in vitro test system of SENP1. IC50 was calculated. The action mode between JK043 and SENP1 was then observed through the docking model. Results The activity test in vitro showed that chemically synthesized JK043 could inhibit the activity of SENP1 and the valued of IC50 was 1.339 μmol/L. The docking model indicated that JK043 interacted with 465W, 468D, 529H, and 597Q of SENP1 protein. Conclusion JK043 has been identified and synthesized, which can inhibit the activity of SENP1 in vitro to a certain extent.