The invention discloses a primary isolation method of animal skin fibroblasts. Disinfected animal skin is selected, and a full-layer skin wound is formed; an injured skin area is taken down after the skin is injured within 3-5 days, subcutaneous tissue and extra blood vessels are removed completely, and the skin is cleaned with PBS (phosphate buffer saline) containing 2% of a penicillin-streptomycin solution for 2-3 times; the skin is cut into tissue pieces ranging from 0.1 mm<3> to 0.5 mm<3> in size; PBS containing 1% of the penicillin-streptomycin solution is poured, centrifugation is performed at the low speed of 1,000 rpm/min for 5 min, and residual floating tissue pieces and a residual supernatant are removed; a cell culture dish covered with 0.2% of gelatin is evenly inoculated with centrifuged tissue pieces, a cell culture fluid containing 2 ml of a DMEM (Dulbecco modified eagle medium), 10% of the FBS and 2% of the penicillin-streptomycin solution is added, and the culture dish is placed in a 5% CO2 incubator at the temperature of 37 DEG C for culture. A skin injury healing mechanism is used, a proper healing period is selected, a large number of proliferative fibroblasts are obtained, meanwhile, removal of the subcutaneous tissue facilitates climbing out of the fibroblasts of corium layers, the number of cells can be increased, the cells can climb out more easily, the cell climbing-out cycle is shortened, and the adherence efficiency is enhanced.