Objective To study the active chemical constituents in Alismatis Rhizome 95% ethanol extract inhibiting CYP1B1 enzyme. Methods The inhibition activity of Alismatis Rhizome ethanol extract toward CYP1B1 enzyme was tested through enzyme incubation reaction experiment. Chemical compositions of Alismatis Rhizome ethanol extract were identified by UPLC-IT-TOF/MS with ESI ion and Shimadzu Composition Formula Predictor software. Results Alismatis Rhizome ethanol extract showed the most significant inhibition with the inhibitory value of 93.96% toward CYP1B1 enzyme. Through the analysis of the multistage tandem mass spectrometry, combined with the application of nitrogen rule and the Scifinder database search, 25 protostane-type triterpenes were identified. Conclusion This study quickly and accurately analyzed the protostane-type triterpenes in the Alismatis Rhizome ethanol extract, which provided the basis for the pharmacodyamic material basis and quality control.