Abstract: The factor of alfalfa protoplasts isolation and culture were discussed, which could provide a basis for the establishment of protoplast culture system. Using cotyledons, hypocotyls and hypocotyl callus of Medicago sativa L.‘Xinmu No.4’as tested materials, the effects of pH value of enzyme, enzymolysis time, enzyme combination, different culture methods on protoplasts isolation and culture were studied. The results showed that when pH value of enzyme was 5.8, the yield of viable protoplast was the highest, at the same time, cell debris was fewer. The optimum enzymolysis time of cotyledons, hypocotyls and hypocotyl callus was 10 h. The enzyme concentration was 2.0% cellulase 0.5% pectinase 0.3% (or 0.5%) macerozyme, the yield of viable protoplast was relatively high. Using liquid medium and solid-liquid double layer medium, protoplast division was observed, but solid-liquid double layer culture was more favorable for protoplast division and culture of Medicago sativa L.‘Xinmu No.4’