Immortalized cell lines with many advantages are widely used in various experimental contexts by many different labs. However, the absence of available cell lines poses difficulties for research in some species, such as camels. To establish an immortalized Bactrian camel fibroblast (iBCF) cell line and understand its biological characteristics, primary fibroblast cells from Bactrian camels were isolated and purified using enzymatic digestion in this study, and telomerase reverse transcriptase (hTERT) vectors were introduced into primary BCF (pBCF) for continuous passage to 80 generations after screening with G418. The cell morphology of different generations was examined under a microscope. Cell cycle and viability were evaluated by flow cytometry and CCK-8 assay, respectively. Cellular genes expression was monitored by qPCR, immunofluorescence, and Western blot, respectively. Chromosomes were determined by karyotyping. The results showed that like most other cells, both pBCF and iBCF were sensitive to nutrient concentrations and adapted to culture in the medium with 4.5 g/L glucose and 10% fetal bovine serum (FBS) concentration. hTERT gene was introduced and stably expressed in iBCF cells, which promoted BCF cell immortalization. The fibroblast specific marker vimentin (VIM) is expressed in both pBCF and iBCF, but epithelial marker cytokeratin18 (CK18) expression is weak in BCF cells. Proliferation and viability detection showed that hTERT-induced iBCF exhibits faster growth rates and higher viability than pBCF. Karyotyping showed that iBCF maintained the same number and morphology of chromosomes as the pBCF. This study demonstrated that we have successfully constructed an immortalized Bactrian camel fibroblast cell line, which was named BCF23. The establishment of the BCF23 cell line provides a foundation for expanding camel-related research.