Abstract: Chitooligosaccharides has a variety of bioactivities due to its special structure features. Currently, almost all of chitooligosaccharides used in activity research were mixtures of polymers with different degrees of polymerization ( DP) . In this study, the specific chitosan enzyme was used to degrade chitosan to prepare chitobiose and chitotriose. The TLC analysis was then used to determine the end point of enzymatic hydrolysis reaction, and the component of hydrolysis products were analyzed by HPLC.The results showed that the enzymatic hydrolysates consisted of chitobiose ( 65.63%) and chitotriose ( 32.48%) .Then, chitobiose and chitotriose were separated by a home-made cation ion exchange resin ( QY-H003) with hydrochloric acid solutions at different concentrations ( C₁= 1.25 mol/L, C₂= 1.55 mol/L) .Further analysis was developed by TLC, HPLC, FT-IR and1 HNMR.The purity of separated chitobiose and chitotriose were 98.06% and 96.00%, respectively. The recovery rate was92.46% and 95.53%, respectively. It indicates that the preparation process was suited for scale production of chitobiose and chitotriose with high purity, and it benefits for the future bioactivity studies and applications of chitooligosaccharides.