The protoplasts were isolated by enzymolysis method from calli of 4 cultivars of Medicago varia (‘Gannong 1’) and Medicago sativa (‘Gannong 4’,‘Algonquin’ and ‘Qingshui’) which have an excellent growth adaptability in Loess Plateau of northwest of China,and the yield and viability of these protoplasts were measured under the different enzymolysis conditions,and their influence factors,such as enzymolysis time,enzyme combination,mannitol concentration,pretreatment measures and subculture time were discussed.Results show that:(1)The optimum pretreatment measure was 0.55 mol/L sucrose or CPW solutions plasmolysis for 1 h and the optimum subculture time was 12 d for 4 cultivars.(2)The best enzyme solution combination for ‘Gannong 1’,‘Gannong 4’ and ‘Qingshui’is 2% cellulose onozuka R-10+0.5% pectinase Y-23+0.3% driselase and for ‘Algonquin’ is 2% cellulose onozuka R-10+0.5% pectinase Y-23+0.3% hemicellulose+0.3% macerozyme+0.3% driselase.Enzymolysis 12 h is best time for ‘Gannong 1’ and ‘Algonquin’,14 h is for‘Gannong 4’and 10 h is for‘Qingshui’.(3)The suitable mannitol concentration was 0.75 mol/L for ‘Gannong 1’,0.65 mol/L for ‘Gannong 4’,0.6 mol/L for ‘Algonquin’ and 0.55~0.6 mol/L for ‘Qingshui’.(4)The regenerated calli can be formed from protoplasts of 4 alfalfa cultivars both by liquid medium and solid-liquid medium,and solid-liquid double layer culture is more helpful for division and regeneration of 4 cultivars than thin layer liquid culture.