Abstract: The enzymolysis peptide of peony flower was studied herein to promote the development and utilization of this flower. The molecular weight distribution and amino acid composition of the peptide were analyzed, and the beneficial effects of the peptide were studied by assessing their ability to inhibit tyrosinase activity, advanced glycation end products (AGEs) formation, and hyaluronidase in vitro. The in vitro results were further verified in a zebrafish melanin accumulation model, results indicated that the active peptide was a small molecule comprising 15 amino acids, with an average molecular weight of 644 Da. At 10 mg/mL, the respective in vitro inhibition rates for tyrosinase activity, AGEs formation, and hyaluronidase were 88.96, 90.50, and 60.43%, respectively. The respective DPPH (2,2-Diphenyl-1-picrylhydrazyl) and hydroxyl free radical scavenging rates were 74.77% and 94.74%, respectively. The zebrafish model studies revealed a melanin inhibition rate of 40.59% when the sample concentration was 1000 μg/mL. In conclusion, the results of these in vivo and in vitro studies preliminarily indicate that the peony flower enzymolysis peptide can potentially improve skin health.