[Objective] The present study aims to provide a basis for in-depth study of the biological characteristics of White Horse (Equus caballus) and the establishment of species resource. [Methods] In this study, using the male and female Ujimchin White Horse in Inner Mongolia as experimental materials, the primary lineage of ear tissue fibroblasts was established by tissue block adherent culture method, the cell adherent rate, survival rate and growth curve of ear tissue source were studied, and the karyotype map of Ujimchin White Horse was further generated. Data results are expressed as Mean ± SD, Excel was used for data collation and analysis, and all graphs were drawn using GraphPad Prism 5. [Results] The results showed that female and male ear tissues of Ujimchin White Horse fibroblasts were cultured by tissue adhesion and grew into a typical fibroblast morphology, and showed the growth characteristics of “S”. There was significant difference in cell survival rate before and after cell freezing (Fig. 5), but the cells still show good resistance to freezing. According to the karyotyping analysis, the number of chromosome strips of female and male Ujimchin White Horse hair tissue fibroblasts is 2n = 64, of which 31 pairs of autosomes and 1 pair are sex chromosomes. [Conclusion] In this study, the fibroblast cell lines of female and male Ujimchin White Horse hair tissues were successfully established, and the cell lines with good genetic characteristics were obtained and stably cultured, which laid the foundation for subsequent in-depth research.