The invention belongs to the technical filed of fat transplantation, and particularly relates to a fat graft rich in high activity fatty granule cells and adipose-derived stem cells and a preparationmethod and application of the fat graft. The method comprises the following steps of performing manual negative-pressure liposuction with an injector, performing centrifugation on obtained fat to obtain a fat layer, enabling the lower layer of the fat layer to be digested to prepare cell suspension, pushing the upper layer to obtain an intermediate layer for transplantation, and finally, uniformlymixing the cell suspension with the intermediate layer to obtain the fat graft rich in high activity fatty granule cells and adipose-derived stem cells. The fat graft has the advantages that comparedwith conventional SVF-gel, the fat graft prepared by the method is higher in yield, and contains massive high-activity granule adipocyte, SVF-cells and adipose-derived stem cells, the concentration of the cells is 5-10 times of that of normal fat tissues, the survival rate of the transplantation fat can achieve 70%, the transplantation fat is soft and uniform in texture, and the probability of adverse reactions of oily tumor masses, tumor tubercles and the like is notably reduced.