Objective To investigate and analyze the association between serum microRNA?141?3p （miR?141?3p） and miR?223?3p levels and neonatal necrotizing enterocolitis. Methods Eighty neonates with necrotizing enterocolitis admitted to our hospital between January 2019 and January 2022 were enrolled as subjects. According to the disease stage， they were divided into the stage Ⅱ group （n = 43） and the stage Ⅲ group （n = 37）. Additionally， 80 healthy newborns born during the same period were recruited as the control group. General clinical data were collected and analyzed systematically. The expression levels of miR?141?3p and miR?223?3p were detected using quantitative real?time PCR （qRT?PCR）. Spearman′s correlation analysis was performed to evaluate the relationship between miR?141?3p， miR?223?3p， and the severity of the disease. Multivariate logistic regression analysis was conducted to assess the impact of miR?141?3p and miR?223?3p on the severity of pediatric illness. Furthermore， a Receiver Operating Characteristic （ROC） curve was plotted to determine the diagnostic value of miR?141?3p and miR?223?3p in predicting the severity of the patient′s condition. Results Compared with the control group， the relative expression levels of miR?141?3p and miR?223?3p in the diseased group were significantly decreased （P < 0.05）. Similarly， compared with the stage Ⅱ group， the relative expression levels of miR?141?3p and miR?223?3p in the stage Ⅲ group were also markedly reduced （P < 0.05）. Furthermore， miR?141?3p and miR?223?3p exhibited a negative correlation with disease severity （r = -0.489， -0.496， P < 0.05）. Increased relative expression of miR?141?3p and miR?223?3p was identified as a protective factor influencing disease severity in children （P < 0.05）. The AUC values for diagnosing the severity of necrotizing enterocolitis using miR?141?3p， miR?223?3p， and their combination were 0.806， 0.783， and 0.885， respectively. Combined diagnosis demonstrated significantly better performance than miR?141?3p alone （Z = 2.050， P = 0.040） or miR?223?3p alone （Z = 2.184， P = 0.029）. Conclusion In neonates with necrotizing enterocolitis， the relative expression levels of serum miR?141?3p and miR?223?3p are significantly decreased， which are closely associated with disease progression and provide potential auxiliary diagnostic value for assessing disease severity.