The invention discloses an in-vitro culture method of chondrocytes, which comprises the following steps: cutting off cartilage tissue pieces from the surface of a joint, and repeatedly cleaning in PBS(Phosphate Buffer Solution) added with double antibodies; putting the cleaned cartilage tissue into a culture medium, cutting the cartilage tissue into pieces, and fully washing the cut cartilage tissue with PBS containing double antibodies; collecting chopped cartilage tissues, centrifuging the tissues, adding a digestive solution, and digesting the tissues; centrifugally collecting cells, adding PBS to clean the cells, adding a culture solution into a cell cluster to prepare a cell suspension, counting the cells, and adjusting the cell density; inoculating the cell, adding a low-sugar DMEMculture medium of FBS, and culturing the cells in a saturated humidity incubator; and when the cells are fused and 80-90% of the bottom of the bottle is covered, carrying out subculture. According tothe invention, the defects and defects of low cell viability, small separation quantity and poor storability in the prior art are solved to the greatest extent, and the mammary epithelial cells cultured by the method have higher cell viability and more quantity and are easier to adhere to the wall compared with the cells obtained by the traditional laboratory method.