An efficient protocol for plant regeneration was developed from protoplasts isolated from calli of Lycium ruthenicum, and the genetic stability of the regenerated plants was analyzed by ISSR and FCM techniques. The results showed that: (1) leaf calli were the best material for producing protoplasts, and in the enzyme solution containing 0.5 mg·mL^-1 mannitol, the yield of protoplasts of leaf calli subcultured once was 7.77×10⁶ pieces·g^-1, vitality was 92%. (2) Improved MS mediumSolidLiquid Double layer culture (MS₂SolidLiquid Double layer) was the best way to culture protoplasts. The frequency of protoplast division was 45.9% after 10 days of culture, and the frequency of cell mass formation was 22.9% after 20 days of culture. (3) In 1.5 mg·mL^-1 6BA+0.1 mg·mL^-1 IBA+MS medium, the protoplasts produced by leaf calli could differentiate into regenerated plants. (4) ISSR showed that the average genetic similarity coefficient of the regenerated plants was 0.88; FCM showed that both the regenerated plants and the parent plants were diploid. The results of this study provide a scientific basis for further research on the somatic hybridization technology of L. ruthenicum to transfer the genetic traits of stress resistance of wild plants, and lay a foundation for the selection and breeding of excellent varieties of L. ruthenicum.