Reported in this paper is a practical method to synthesize quaternary ammonium salt of chitosan (HACC) in high substitution rate by reacting chitosan with 2,3-epoxypropyltrimethyl ammonium chloride. Reaction conditions were screened and the main factors were analyzed independently. Optimized condition factors were determined as follow: mole ratio of the reactants nc:ng = 1:4; molecular weight of Chitosan MWc = 320000g/mol; alkaline pretreatment time ta = 14h; moisture content of the pretreated chitosan Wi = 20%; reaction pH = 7; reaction temperature T = 75℃; reaction time tr =8h. The substituted product was analyzed by IR, SEM and TG/DTG. The analysis results indicate that the substitution occurred mainly on the amino groups of chitosan. Compared to unsubstituted chitosan, the substituted product has a smaller particle size and slightly lower thermostability.