N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC) hydrogel microspheres were prepared by premix membrane emulsification and thermal gelation methods, using HTCC/a-b-glycerophosphate mixed system with the property of thermal gelation as water phase. The effects of preparation conditions on size distribution, structure and drug encapsulation efficiency of microspheres, including transmembrane pressure, formulation of water phase and oil phase, volume ratio of water to oil phase and pore size of microporous membrane, were examined. After optimization, uniform-sized microspheres (polydispersity, PDI<0.1) with different sizes and high drug encapsulation efficiency (75.49%±2.62%) were obtained. The cytotoxicity and pH-sensitivity of as-obtained hydrogel microspheres were evaluated in comparison with the microspheres prepared by traditional method. The results showed that the as-obtained hydrogel microspheres possessed good biocompatibility and outstanding pH-sensitivity, and remained stable in neutral/alkalescent media and released drug in a relatively slow manner, with the drug cumulative release rate of 34.6% at 24 h in buffer solution at pH=7.4. While the hydrogel microspheres collapsed in acidic solution and led to a repaid drug release, and the cumulative release rate reached up to 79.6% in 1 h in buffer solution at pH=5.5.