Abstract: Hair follicles were isolated from goat ear skin by 2.4 U/mL Dispase digestion and the bulges were separated with special fine needle and digested with 0.5 mg/mL trypsin plus 0.2 mg/mL EDTA. The resultant cells were cultured in self-prepared serum-free conditional medium and studied through morphological observation, cell growth curve and colony-forming efficiency(CFE), and identified by immunohistochemical staining. The results showed that the cells were slide, small-sized, ball-shped, obviously threedimensional and undifferentiated, immunohistochemically positive to K19, integrin-β1 and p63 staining and so performed stem cells attributes and high CFE. The CFEs of the third, the fifth and the seventh passages were respectively 25.6%±2.6%, 31.8%±1.9 % and 27.0%±3.9%, significantly higher than that of control (P<001). And the data proved that the goat follicle stem cells could be subcultured for 19 times in vitro in the serum-free conditional medium self-prepared.