Necrotizing enterocolitis (NEC) in preterm infants is an often fatal, gastrointestinal emergency. There is a need for a robust NEC biomarker that can be adopted in pathology labs and that is not confounded by sepsis. Use of such a NC biomarker could improve bedside management, lower morbidity and mortality, and permit patient selection in clinical trials for therapeutic candidates. We evaluated if aberrant intestinal alkaline phosphatase (iAP) biochemistry in infant stool is a molecular biomarker for necrotizing enterocolitis and not correlated to sepsis in the absence of NEC. There were 136 premature infants enrolled in three hospitals in this observational and prospective study. Median birthweight (IQR) was 1050 g (790‐350 g) and median gestational age was 28.4 weeks (26‐0.9 wks). Twenty‐five infants were diagnosed with severe NEC, 19 were suspected of having NEC, and 91 were non‐NEC infants. Twenty‐six patients were diagnosed with late‐onset sepsis and 14 had other non‐GI infections. Infant stool samples were collected between 24–40+ weeks postconceptual age (PCA). Enrolled infants were subject to abdominal radiography at physician and hospital site discretion. Enzyme activity and relative abundance of iAP were measured using fluorometric detection and immunoassays, respectively. Post‐measurement, biochemical data were evaluated against clinical entries from infants’ hospital stay. Fecal iAP content was 99±19, 123±41, and 5±3 (median±SE) for severe, suspected, and non‐NEC samples, respectively. Enzyme activity was 183±108, 355±180, and 613±218 kU/g (median±SE) for severe, suspected, and non‐NEC samples, respectively. ROC area under the curve (AUC) values were 0.96, 0.97, 0.54, and 0.58 for relative iAP content measurements at time of severe NEC, suspected NEC, sepsis, and other non‐GI infections; AUC values for iAP activity were 0.60, 0.76, 0.52 and 0.57, respectively. High amounts of iAP protein in stool and low iAP enzyme activity are reliable and specific biomarkers for NEC: iAP biochemistry uniquely has clinical utility in distinguishing NEC from sepsis.Support or Funding InformationThis work was supported by the National Institutes of Health (R41HD095779 to RB and SK; R01GM097350 to SK; K08DK101608, R03DK111473 and R01DK118568 to MG), National Science Foundation (IIP‐1713220 and IIP‐1547932 to RB and SK); March of Dimes (5‐FY17‐79 to MG), Louisiana Board of Regents (LEQSF‐RD‐D‐07 to SK), LSU LIFT2 (HSCNO‐2017‐LIFT‐006 to SK), Children’s Discovery Institute of Washington University and St. Louis Children’s Hospital (MG), Department of Pediatrics at Washington University School of Medicine, St. Louis (MG), LSU School of Medicine (BB, SK, and MH), and LSU Health Foundation (MH, ZG, and SK).