Abstract: Botryosphaeria dothidea causes dry rot disease in Carya cathayensis, resulting in significant economic losses. To obtain a large number of high-quality protoplasts, the optimal conditions for the preparation protoplasts were explored, using the enzymolysis method. The results demonstrated that B. dothidea was cultured in PDA medium for 48 h with a mixed enzyme solution containing cellulase, snailase, and lyticase, yielding the maximum protoplasts in a situation where the concentration of total enzyme was 5 mg·mL⁻¹, the enzymatic time was 5 h, and the enzymatic temperature was at 35 ℃. In this study, the enhanced green fluorescent protein (EGFP) was introduced into protoplasts using polyethylene glycol (PEG)-mediated method, followed by comprehensive observation and analysis of the transformed protoplasts. There were no significant differences between transformed strains and wild-type strains in terms of PCR detection of the gene, fluorescence microscopy observation, colony morphology, mycelial growth rate and pathogenicity assays and the like, which indicated the successful integration of the EGFP gene into the strain. The research not only optimized conditions of protoplast preparation, but also validated the effectiveness of the transformation system by observing the transformed protoplasts, thereby laying a foundation for further investigation into the pathogenesis of dry rot disease in C. cathayensis and the screening of disease-resistant cultivars.