The effects of chitin pretreatment with four different solvent systems (viz., sodium hydroxide/urea, phosphoric acid, lithium chloride/N,N-dimethylacetamide, and choline chloride/thiourea) on the structure of the polysaccharide and the efficiency of its enzymatic deacetylation were investigated. Changes in the chitin structure before and after pretreatment were characterized using scanning electron microscopy (SEM), Fourier-transform infrared (FT-IR) spectroscopy, and X-ray diffraction (XRD). The pretreated polysaccharide was used as the substrate for enzymatic reactions, with chitin deacetylase derived from Colletotrichum lindemuthianum used as the biocatalyst. The effects of the different pretreatment methods on the enzymatic deacetylation of chitin were systematically analyzed. Although the four pretreatment methods exerted a negligible effect on the degree of deacetylation (DD) of chitin, they significantly altered its crystallinity and molecular weight. Structurally, the decreases in chitin crystallinity and particle size enhanced the efficiency of its enzymatic deacetylation. The activity of chitin deacetylase was increased by 121%~293% depending on the chitin pretreatment method used. Chitin pretreated with sodium hydroxide/urea was identified as the optimal substrate, with its DD increased by 4.73-fold compared with that of untreated crystalline chitin. This study demonstrates that the pretreatment of crystalline chitin with sodium hydroxide/urea is an effective strategy for improving the efficiency of its enzymatic deacetylation, thus providing new technological insights and a theoretical foundation for the enzymatic production of this important polysaccharide.