In order to study the effect of venturi tube-based hydrodynamic cavitation on the ionic cross-linking preparation of chitosan microspheres and the encapsulation of methylisothiazolinone (MIT), which was used as a model antibacterial agent in this study. The effects of inlet pressure of venturi tube，cavitation time, chitosan concentration, mass ratio of chitosan and sodium tripolyphosphate (TPP), MIT concentration on the entrapment efficiency of chitosan microsphere were investigated, and the entrapment efficiency of microsphere prepared by venturi tube-based cavitation was compared with that prepared by conventional mechanical agitation. The results showed that the chitosan microspheres could be stably obtained by using venturi tube-based cavitation when the mass ratio of chitosan and TPP was larger than or equal to 2.5. The optimum technological conditions were as follows: the inlet pressure of venturi tube was 0.2 MPa, cavitation time was 20 min, chitosan concentration was 3.0 g/L, TPP concentration was 3.0 g/L, MIT concentration was 0.5 mmol/L, and the entrapment efficiency could be up to 62.3%. Compared to that prepared by conventional mechanical agitation, the entrapment efficiency of microsphere prepared by venturi tube-based cavitation was increased by at least 17.7%.