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This invention relates to the field of biomedical technology and provides a controllable cryogenic treatment mesenchymal stem cell preparation, its preparation method, and its application. The method includes the following steps: cell preparation and cryopreservation equilibration: digestion and collection; cryopreservation equilibration; removal of cryoprotectant and washing: centrifugation to remove cryopreservation solution; PBS washing; cryogenic incubation: resuspending the final cell pellet after multiple washes in pre-cooled PBS; incubating the cell suspension at 4±0.5℃ for 72 hours±10 minutes; after incubation, removing the cell suspension from the 4℃ environment, centrifuging, and resuspending in PBS. This invention yields a ready-to-use, easily stored and transported, and functionally enhanced RT-MSCs preparation. Although the cells in this preparation have lost their proliferative and metabolic activity, they still retain the complete therapeutic functions of MSCs, such as immunomodulation, anti-fibrosis, and angiogenesis promotion.