The protonation constants of chitosans with different molecular weight (Mw) and degree of deacetylation were demonstrated by the modified Henderson-Hasselbalch model and Hogfeldt’s three-parameter model. The protonation constants pKa of chitosan showed a slightly decreasing from 6.51 to 6.39 when the molecular weight changing from 1370 to 60 kDa. The degree of deacetylation showed a greatly effect on pKa values, which were increased from 6.17 to 6.51 with the degree of deacetylation decreasing from 94.6 to 73.3%. The degree of deacetylation influenced the balance of hydrophobic interactions and hydrogen bondings on chitsoan. The fitting of the titration data to both two methods showed that Hogfeldt’s three-parameter model provided a better agreement. The results obtained in this paper allowed to a better understanding of some physicochemical mechanisms and biological properties of chitosan.