Background: Necrotizing enterocolitis (NEC) is a severe inflammatory gastrointestinal disease that affects premature neonates with high morbidity and mortality. We aimed to evaluate the potential of IgG anti-tissue transglutaminase antibodies (IgG tTG) as biomarkers for NEC and to explore their applicability in the early diagnosis, monitoring prognosis. Method: We conducted a prospective observational study on 60 neonates with abdominal distension, dividing into the NEC(n=30) and the control(n=30) groups according to the follow-up results. We collected plasma samples within 48 h of the onset of abdominal distension, and used the autoantigen microarray to screen for NEC-associated autoantibodies. Additionally, an Enzyme-linked immunosorbent assay (ELISA) was utilized to measure the levels of IgG tTG in a validation study that included 43 neonates with NEC and 20 gestational age- and weight-matched controls. Results: The autoantibody microarray analysis indicated that plasma levels of IgG tTG were significantly higher in neonates with NEC compared to controls ( P< 0.001). ELISA confirmed the significant elevation of plasma IgG tTG in neonates with NEC ( P<0.001). Plasma IgG tTG were able to distinguish NEC from the control group, with an area under the curve (AUC) of 0.8674 (95% confidence interval (CI): 0.7794 - 0.9555, sensitivity of 72.09% and specificity of 95%). Encouragingly, IgG tTG levels were significantly higher in NEC stage I than in the controls ( P < 0.001). Furthermore, as neonates with NEC showed clinical improvement, the levels of IgG tTG decreased ( P=0.0159). Conclusion: IgG tTG may serve as a biomarker for diagnosis early NEC and predict its prognosis.