This study amied to explore the culture method of biliary tract fibroblasts from Diannan small-ear pigs, and lay the foundation for further exploration on biliary tract benign stenosis. The biliary tract injury model was constructed via adopting Diannan smallear pigs, and the tissue adherence method was adopted. After trying different medium and fetal bovine serum concentrations, the culture method of mature Diannan small-ear pig biliary tract fibroblast was explored. Additionally, vimentin and desmin was examined and identified as fibroblasts. The biliary tract fibroblasts that can be passaged were available to be cultured via tissue adherence method. Biliary tract fibroblast, a sort of cell with proliferation and multidirectional differentiation potential, is difficult to be cultured and passed on, and its successful culture lays a good foundation for further exploration on drug intervention.