Abstract: This study utilized cladodes of Asparagus cochinchinensis to investigate the effects of enzyme concentration and hydrolysis time on protoplast isolation. A dual-factor orthogonal experiment was subsequently conducted to optimize the isolation protocol. The results demonstrated that optimal conditions, consisting of 1.5% Cellulase, 0.8% Macerozyme and 3 h of hydrolysis, yielded the highest values for both total protoplast count and viable protoplast numbers. This provides important theoretical support for efficient protoplast isolation from A. cochinchinensis.