Objective: To investigate and improve the culture method of rabbit articular chondrocyte using autoserum in vitro. Methods: The 3-month New Zealand rabbit was killed to obtain the autoserum, 4-week New Zealand rabbit cartilage of bilateral knee was resected under aseptic condition. Chondrocyte was isolated by type Ⅱcollagenase enzyme digestion and mechanical isolation method. The cell was cultured and passaged using autoserum, then identified by morphologic observation, toluidine blue staining, and type Ⅱ collagen enzyme immunohistochemical, growth curve was pictured by MTT method. Results: It showed that the primary cultured chondrocyte adherented after 2 h under the Inverted microscope, and formed monolayer formation after 8 h. The chondrocyte was ready to be passaged after 24 h cultivation. The first to the fifth generation chondrocyte phenotype was stable, and the proliferation ability was well. Toluidine blue staining showed that the nucleus of cultured chondrocytes were blue and cytoplasm was pale blue; immunofluorescent staining showed that cultured chondrocytes had a positive expression of collagen type Ⅱ that the color was tawny; proliferative rate of chondrocytes were detected by MTT showed that the OD value in the first to fifth generation had no differences (P<0.05). Conclusion: The research indicated that the first to the fifth generation chondrocyte was used to the research of knee osteoarthritis.