In this study, a synergistic catalytic system of chitinase (ChiA) and β-N-acetylhexosaminase HJ5N that can degrade colloidal chitin to prepare N-acetylglucosamine (GlcNAc) in one-step reaction was established. The effects of different factors on the catalytic efficiency and the mechanism of the synergistic action of the enzymes were discussed. The results showed that the optimum reaction conditions were 30 ℃, pH 6 and concentration ratio ChiA to HJ5N of 2:1. Under these conditions, 17.55 mg/mL of GlcNAc was produced with a yield of 58% when 90 g/L of (containing 30.3 g/L of colloidal chitin) was used as the substrate. HJ5N could ensure quick conversion of the intermediate product (GlcNAc)₂ into GlcNAc, thus removing the inhibitory effect of (GlcNAc)₂ on ChiA and ultimately allowing the efficient degradation of colloidal chitin into GlcNAc. This study provides an effective strategy for the industrial application of enzymatic degradation of colloidal chitin to prepare N-acetylglucosamine.