The invention discloses a method for preparing an isolated hair follicle in a process of treating scar by hair follicle stem cell transplantation. The method comprises the following steps: extractionof hair follicles, separation of the hair follicles, completely separating the hair follicle units into single hair follicles under an electron microscope, and keeping the extracted skin appendages such as sebaceous glands, sweat glands and the like as far as possible; for scars in a smooth hairless area, removing lower 1/3 of hair papilla, so that the hair follicle could not enter the next growthcycle, and no final hair grows in the scar area; in vitro culture of the hair follicles, putting the isolated single hair follicle into a special culture solution for culturing for later use; planting the hair follicle stem cells in a specific area of the scar on the basis of hair follicles. According to the method, through a hair follicle in-vitro culture technology, the single hair follicle isseparated and inactivated by adopting a separation method different from a hair transplantation operation, and is cultured in the special culture solution, so that growth and proliferation of hair follicle stem cells are facilitated. The method can achieve purposes of inhibiting scar hyperplasia, softening and repairing scars, and removing and beautifying scars.