Encapsulating proteins in polymeric microspheres is a useful mode of drug delivery, but the proteins are subjected to damage in the process of ultrasound emulsion microencapsulation. The objective of this study was to investigate the effects of ultrasound power and duration on the function and structure of trypsin, and the reason of protein denaturation when it was irradiated by 20 kHz ultrasound. The relatively stable enzyme, trypsin, was dissolved in aqueous solution in the presence and absence of additives to study the stability of trypsin during the ultrasound irradiation. The damage of the molecular structure of trypsin was detected via combined high performance liquid chromatogram and electrospray ionization mass spectrometry (HPLC-ESI-MS). The results showed that the activity of trypsin decreased with increasing ultrasound power from 100 to 500 W or extending the irradiation time from 1 to 20 min. This effect could be enhanced via aerating the solution for a duration 10 min at 300 W. Fragments of trypsin were detected in the treatment (300 W, 10 min) by HPLC-ESI-MS. The additives, Tween 80 and mannitol, could protect trypsin against the inactivation caused by ultrasound. The reason of inactivation was partly from the alteration of the molecular conformation and partly from the modification or damage of trypsin's molecular structure.