Abstract: Objective： To explore the methods of isolation, purification, and identification of intrahepatic cholangiocarcinoma (iCCA) associated fibroblasts (iCAFs), which could facilitate further functional and pharmacological evaluation. Methods： Under aseptic conditions the tissue specimens of iCCA were obtained, minced, and digested in the digestive medium supplemented with collagenase Ⅳ and hyaluronidase. The single-cell suspension underwent immunomagnetic separation and purification, and carcinoma-associated fibroblasts (CAFs) were separated and cultured. Through morphological observation, immunofluorescent staining and flow cytometry analysis, whether the cells were iCAFs or not. Results： Morphologically, iCAFs were elongated or fusiform, with transparent cytoplasm, obvious refraction, single round or ovoid nucleus. Cellular processes overlap to form grids and show the "peak-valley" pattern. Under immunofluorescence, the typical iCAFs showed positive staining for alpha smooth muscle actin (α-SMA) and vimentin, and negative staining for pan-cytokeratin. Under flow cytometry detection, they were positive for α-SMA and negative for both CD34 and CD45. Conclusions： Enzyme digestion in combination with immunomagnetic separation can effectively separate iCAFs with high purity, providing a good platform for further studies on the roles of iCAFs in the pathogenesis of iCCA.