The invention provides human skin fibroblast and a preparation method thereof, and relates to the medical field of stem cells and regeneration. The preparation method comprises the following steps ofperforming pretreatment: collecting retroauricular skin tissue, separating dermis layer tissue, performing thorough cleaning with tissue cleaning liquid, and performing shearing to obtain tissue broken pieces; performing digestion for the first time: mixing the tissue broken pieces with tissue digestive juice I, performing digestion, after digestion, performing centrifuging, removing supernatant,and reserving precipitate, wherein the tissue digestive juice I is normal saline containing neutral protease and deoxyribonuclease; performing digestion for the second time: mixing the precipitate reserved after the digestion for the first time with tissue digestive juice II, performing digestion, adding trypsin, and continuing digestion, wherein the tissue digestive juice II is DNEM culture mediums containing I type collagenase and deoxyribonuclease; ending digestion: adding a stopping solution, ending the digestion, screening out tissue pieces, performing centrifuging, removing supernatant,and reserving precipitate; and performing culturing: re-suspending the precipitate with selective culture mediums, performing subpackaging and performing culturing. The cells obtained by the preparation method disclosed by the invention are large in quantity, high in purity and good in activity.