Skin was the first barrier to protect fish body from the out-environment, and was the main carrier of the body color. For lack of fish skin cell lines, investigation on skin function and color regulatory mechanism reached a plateau. In this paper, Rhodeus sinensis was used to investigate the fish skin cell separation method, with modified enzyme digestion ways. Rhodeus were kept in bacteriostasis and fungistasis solution for about 8-24 hours. After digesting in the 0.25% Trypsin-EDTA for 3-5 min, Rhodeus were killed and stroked off the scales. Peeled fish skin, and removed muscle or other tissue which jointed to the skin in stereo microscope. Then skin was digested by collagenase IV and trypsin jointed digestion method. Later, Rhodeus Skin Derived Cells, RSDCs for short, were collected and cultured at 28℃, 5% CO₂. The growth curve was drawn by hemacytometer counting method. The expression of epithelial markers, Keratin 18 & Vinculin A, and endothelial label gene, collagen I, were tested by RT-PCR method at F₀, F₅ and F₁₀ generation. RSDCs lines were obtained and cultured well with doubling time of 30 h and "S"-growth-curve, which was familiar with the other adherent cells. The RT-PCR results revealed that the expression of Keratin 18 and Vinculin A declined along with cell passage cultivation, while the expression of Collagen I was on the rise. Our researches indicated that modified enzyme digestion method should be ideal method to obtain fish skin cell. The obtained RSDCs growth curve was typical adherent cells "S" type. And the obtained RSDCs were derived from epithelium (ectoderm) and endothelium (mesenchyme). Meanwhile, along with passage cultivation, the proportion of epithelium declined.