Objective <p>This study was to investigate the diagnostic and prognostic value of serum homocysteine (Hcy) and activated matrix metalloproteinase-8 (aMMP-8) in acute pancreatitis (AP).</p> Methods <p>A retrospective cohort study was conducted on 285 patients with AP. Using a 1:1 propensity score matching technique, controls were selected for the AP group, leading to 245 pairs being matched (245 AP patients and 245 controls). The severity of AP was assessed using the BISAP score. The prognosis of AP patients within six months was assessed. Spearman’s correlation analysis was performed; ROC curves were used for efficacy analysis; Kaplan-Meier curves were employed for survival analysis; and logistic regression was conducted for risk factor analysis.</p> Results <p>Serum Hcy and aMMP-8 levels were significantly higher in the AP group than in the control group, and there was a moderate positive correlation (<i>r</i> = 0.514). Hcy and aMMP-8 were higher in severe AP than in mild-to-moderate AP and in those with poor prognosis than those with good prognosis. The serum Hcy+aMMP-8 test had high efficacy in diagnosing AP (AUC = 0.982) and predicting poor prognosis (AUC = 0.941). High Hcy and aMMP-8 levels increased the risk of poor prognosis of AP patients within six months. Serum Hcy and aMMP-8 were independent risk factors for poor prognosis within six months in patients with AP. The Hosmer-Lemeshow test indicated good model fit.</p> Conclusion <p>Serum Hcy and aMMP-8 are elevated in AP patients and are closely associated with AP severity and poor prognosis.</p>