Objective To investigate the relationship between serum melatonin and prostaglandin E₂ （PGE2） levels with disease severity and prognosis of necrotizing enterocolitis （NEC） in premature infants. Methods A total of 215 premature infants diagnosed with NEC （NEC group） admitted to our hospital between January 2022 and February 2025 and 80 healthy premature infants （control group） were prospectively enrolled. NEC cases were stratified by Bell′s staging divided into mild （n = 67）， moderate （n = 90）， and severe （n = 58） subgroups. Serum levels of melatonin and PGE2 were measured by enzyme-linked immunosorbent assay. Spearman correlation analysis was used to assess the relationship between serum melatonin， PGE2 levels and Bell staging. According to the 28-day prognosis assessment， NEC infants were divided into a poor prognosis group and a good prognosis group. Multivariate logistic regression and ROC analysis were used to explore the association and predictive value of serum melatonin and PGE2 levels in NEC prognosis， and decision curve analysis （DCA） was used to evaluate clinical net benefit. Results Compared with the control group， the NEC group had significantly lower serum melatonin levels and higher PGE2 levels （P < 0.05）. Among the NEC subgroups， serum melatonin levels decreased and PGE2 levels increased progressively from mild to severe NEC （P < 0.05）. Serum melatonin was negatively correlated with Bell stage， while PGE2 was positively correlated （P < 0.05）. The 28-day poor prognosis rate in NEC infants was 18.60% （40/215）. Severe disease and elevated PGE2 were independent risk factors for poor prognosis， while higher birth weight and increased melatonin were independent protective factors （P < 0.05）. The area under the ROC curve （AUC） for predicting prognosis using melatonin， PGE2， and their combination was 0.797， 0.780， and 0.880， respectively， with the combined prediction performing significantly better than either biomarker alone （P < 0.05）. DCA indicated that when the threshold probability exceeded 0.1， the net benefit of the combined model was greater than that of either marker alone. Conclusion In premature infants with NEC， reduced serum melatonin and elevated PGE2 levels are associated with increased disease severity and poor prognosis. The combination of serum melatonin and PGE2 levels provides high predictive value for prognosis.