The present disclosure relates to the cosmetology field, in particular to an activated revitalizing lotion and its preparation method and application. A preparation method of an activated revitalizing lotion comprises the steps of: mixing an isolated blood sample, ozone-oxygen mixture and sodium citrate, and centrifuging the mixed blood sample at 1800-2000 r/min for 9-10 min to obtain a plasma supernatant for use as an activated revitalizing lotion; the ozone-oxygen mixture has an ozone concentration of 42.8-171.4 μg/L and added in an amount of 2.3-3.2 L/1 L of the isolated blood sample; and sodium citrate is added in an amount of 0.2-0.3 mg/1 L of the isolated blood sample. The activated revitalizing lotion prepared by the preparation method of the present disclosure shows improved effect in repair and revitalization of skin or subcutaneous tissues.