Abstract: In this paper, the preparation process of D-glucosamine hydrochloride (GAH) was investigated from the waste residues of Pleurotus eryngii (P. eryngii). Liquid chromatography-mass spectrometry (HPLC-MS), infrared spectroscopy (IR), and physicochemical indexes of GAH were also studied. Then the effect of GAH on the zebrafish embryonic development was evaluated. The experimental conditions for preparing GAH were optimized through single-factor experiments and response surface methodology. Under optimal conditions (HCl concentration of 31%, hydrolysis time of 4 h, hydrolysis temperature of 82 ℃, and the liquid-solid ratio of 5 mL/g), the yield of GAH reached 23.61%. HPLC-MS, IR, and physicochemical index analysis showed that the purity of the GAH-purified sample was 101.9% of the standard. The mass spectrometry and infrared spectroscopy were consistent with the standard. All indexes met or even outperformed the specifications of the United States Pharmacopeia 43-National Formulary 38 (USP 43-NF38), and the arsenic content was only 0.21 μg/g. In addition, the zebrafish experiment indicated that exposure to moderate and high concentrations (0.3%~0.5%) of GAH could induce more than 50% of the abnormal embryonic development of zebrafish. Moreover, 0.3% of GAH promotes the transforming growth factor-β (TGF-β) signal pathway. In conclusion, the results provide valuable insight into preparing and applying the industrial residue of P. eryngii.