Objective To explore the expression of serum prokineticin 2 (PK2) and chemokine cytokine ligand3 (CCL3) in neonates with necrotizing enterocolitis (NEC) and their clinical significance. Methods A total of 127 NEC neonates admitted to Qingdao Women and Children’s Hospital from January 2022 to January 2025 were selected as case group, who were divided into stage I (n=35), stage Ⅱ (n=59), and stage Ⅲ (n=33) according to Bell staging criteria. After 30 days of treatment, the patients were further categorized into good prognosis group (n=82) and poor prognosis group (n=45) based on outcomes. Additionally, 127 healthy neonates were randomly selected as control group during the same period. Serum PK2 and CCL3 levels were measured using enzyme-linked immunosorbent assay. Spearman rank correlation was used to analyze the association between serum PK2, CCL3 and disease severity, while Pearson product moment correlation was employed to examine the association between PK2 and CCL3. Multivariate logistic regression was used to assess the association of these markers with mortality risk. Receiver operating characteristic (ROC) curves were generated to evaluate their diagnostic value for NEC and predictive value for mortality risk. Results Compared with control group, PK2 and CCL3 levels were elevated in case group (P<0.05). The AUCs for PK2, CCL3, and their combination in diagnosing NEC were 0.857, 0.865, and 0.937, respectively, with the combined diagnostic value being superior to that of either marker alone (Z=1.759, 1.643; P=0.017, 0.019). Compared with stage Ⅰ patients, serum PK2 and CCL3 levels were higher in stage Ⅱ and Ⅲ patients, with stage Ⅲ patients showing higher levels than stage Ⅱ patients (P<0.05). PK2 and CCL3 were positively associated with disease severity (rs=0.681, 0.702, P<0.05), and PK2 was positively associated with CCL3 (r=0.734, P<0.05). Compared with good prognosis group, poor prognosis group had higher PK2 and CCL3 levels (P<0.05). High PK2 and high CCL3 were independent risk factors for poor prognosis in NEC neonates (OR=1.914, 1.797; P<0.05). The AUCs of PK2, CCL3, and their combination for predicting poor prognosis in NEC neonates were 0.796, 0.800, and 0.892, respectively. The combined predictive value was superior to that of either single indicator (Z=1.648, 1.579; P=0.019, 0.023).Conclusion Serum PK2 and CCL3 are up-regulated in neonates with NEC, which are closely associated with disease severity and prognosis. Early combined detection may serve as a biochemical marker for auxiliary diagnosis of NEC and prediction of poor prognosis in neonates