【Objective】This study aims to establish an efficient protoplast isolation system for Ziziphus jujuba ‘Dongzao’ fruit. By systematically analyzing the effects of various factors on protoplast extraction, it seeks to provide technical support for subsequent research on gene function and the molecular mechanisms underlying fruit development.【Method】Using semi-red fruits of Z. jujuba ‘Dongzao’ as experimental material, a multi-factor experimental design was applied to optimize key parameters. The factors investigated included: enzyme combination (cellulase, macerozyme, pectinase), KCl concentration (150, 205, 255, 305, 355, 405, 430, 455, 505 mmol/L), enzymatic hydrolysis duration (2.0, 2.5, 3.0, 3.5, 4.0 h), and vacuum (200 Pa) infiltration time (10, 20, 30 min). Through systematic screening, the optimal concentrations of cellulase, pectinase, macerozyme, and KCl were determined, leading to the identification of the most effective enzyme solution for protoplast isolation.【Result】The optimal enzymatic solution consisted of 0.02 g/mL cellulase, 0.01 g/mL pectinase, 0.01 g/mL macerozyme, 430 mmol/L KCl, 65 mmol/L CaCl₂, 10 mmol/L MES (2-morpholinoethanesulfonic acid), and 1 mmol/L DTT (dithiothreitol), adjusted to pH 5.8. The protoplast yield reached 1.82 × 10⁶ /mL when the tissues were subjected to vacuum infiltration at 200 Pa for 20 min, followed by 3.0 h of enzymatic digestion in the dark on a horizontal shaker. Under these conditions, the isolated protoplasts exhibited intact morphology and high viability.【Conclusion】By screening four key factors—enzyme concentration, hydrolysis time, KCl concentration, and vacuum treatment duration—an efficient enzymatic protocol was established for isolating protoplasts with intact cellular structure and high viability from Z. jujuba ‘Dongzao’ fruit. This study successfully develops a robust protoplast separation system, which provides a reliable technical foundation for future genetic functional studies and the molecular analysis of fruit development in Z. jujuba ‘Dongzao’.