Abstract: The aim is to provide materials for ploidy breeding and cell fusion of wild jujube, and to open up a new path for hybrid breeding and variety renewal of jujube. The protoplast was separated by enzymatic hydrolysis from the pollen of wild jujube tetrad. The protoplast yield was calculated by hemocyte counter and the protoplast viability was determined by FDA. The results showed that when mannitol was used as osmotic regulator in the mixed enzyme solution of 1% snail enzyme and 0.5% cellulase, after 4 hours of enzymatic hydrolysis,the isolation rate of Pollen Protoplasts was the highest at the tetrad stage, reaching 4.8×105A/mL, by 0.01% FDA activity, the vitality of Pollen Protoplasts the tetrad stage was 21.1%. Degree of mannitol affected the isolation of pollen protoplasts. In 0.3 mol/L mannitol, the isolation rate of Pollen Protoplasts at tetrad stage was 3.8 ×105A/mL , and the vitality was 27.3%.isolation of Pollen Protoplasts in tetrad stage provides materials for subsequent triploid breeding, and provides practical basis for protoplast isolation in other pollen stages of wild jujube.