Objective To establish a method for primary culture of adult dermal fibroblasts. Methods Fresh skin tissues were digested with dispase II to separate the epidermis from dermis. Then fibroblasts were isolated from the dermis by tissue-adhesive and type I collagenase digestion method respectively. The growth of cells was observed under an inverted phase contrast microscope. the expression of vimentin in cultured cells was detected by immunofluorescence cytochemical staining to identify cell type and assess the purity of fibroblasts. The proliferation activity of fibroblasts was analyzed by MTT assay and drew growth curve. Cell cycle distribution was detected by flow cytometry. Results The fibroblasts were cultured for 48 hours, and more than 95% adherent cells were obtained by collagen protease separation. At the same time, the cells began to grow from the adherent wall at the edge of the tissue block. A large number of fibroblasts adherently grew on day 5. The cells grew rapidly, and on day 7, the layers of proliferating cells were covered in petri dishes. A small number of epithelial cells were mixed in fibroblasts obtained from the tissue-adhesive method, while the vimentin positive rate of cells isolated by collagenase digestion method was nearly 100%, almost all of which were fibroblasts. Conclusion Both collagenase digestion and tissue-adhesive method are rapid, economical and effective methods for obtaining dermal fibroblasts. Collagenase digestion method can prevent epithelial cell contamination more effectively, and the isolated dermal fibroblasts have good proliferative capacity.