Abstract: To investigate the potential of enzymatic hydrolysis for enhancing the nutritional and functional properties of Jinhua ham, a concentrated extract produced by dialysis desalination was employed as a substrate. A comprehensive evaluation process encompassing degree of hydrolysis (DH), peptide yield, and sensory analysis was utilized to select the most suitable protease for hydrolyzing Jinhua ham. Subsequently, an orthogonal experimental design was implemented to optimize the hydrolysis conditions using the chosen protease. Results revealed that trypsin exhibited superior efficacy in hydrolyzing Jinhua ham, achieving optimal hydrolysis under the following conditions: an enzyme-to-substrate ratio of 1600 U/g, pH8.0, substrate concentration of 10%, hydrolysis temperature of 37 °C, and hydrolysis time of 5 h. The optimized hydrolyzed solution achieved a DH of 9.74% and a peptide yield of 40.60%. Evaluation of the antioxidant properties of the hydrolyzed peptides demonstrated significant radical scavenging activity, with IC₅₀ values of 8.02 mg/mL and 4.61 mg/mL against DPPH and ABTS⁺ radicals, respectively. However, the peptides exhibited relatively weak Fe²⁺chelating ability. Furthermore, the hydrolyzed peptides displayed a pronounced activating effect on alcohol dehydrogenase (ADH), achieving an activation rate of 65.62% at a concentration of 1 mg/mL. The molecular weight distribution of the hydrolyzed peptides was dominated by peptides below 2000 Da, accounting for 88.95% of the total peptide content. Liquid chromatography-mass spectrometry (LC-MS/MS) analysis identified a total of 3235 unique peptides in the hydrolyzed Jinhua ham extract. The major source proteins of these peptides were identified as myosin, ATP-binding cassette proteins, and actin. This research provides novel insights into the potential of Jinhua ham as a source of bioactive peptides, paving the way for future exploration and development within the ham industry.