The aim of this study was to determine the changes of the biomarkers used for the diagnosis of necrotising enterocolitis of human neonates in premature calves with respiratory distress syndrome (RDS). Novel biomarkers including the intestinal fatty acid binding protein (IFABP), the liver-type FABP (LFABP), trefoil factor-3 (TFF3), actin gamma 2 smooth muscle (ACTG2), and Claudin-3 were investigated using bovine specific ELISA kits. Thirty premature calves with respiratory distress syndrome (the RDS group), seven premature calves without RDS (the non-RDS group), and seven healthy calves (control) were included in the study. Blood samples from all the groups were taken at 0 and 48 h for the blood gas and biomarker measurement. It was determined that IFABP (P < 0.05), LFABP (P < 0.05), TFF3 (P < 0.05), ACTG2 (P < 0.05), and Claudin-3 (P < 0.05) in the control group were significantly higher than those in the RDS and non-RDS groups at 0 hour. The LFABP and Claudin-3 concentrations in the control group were statistically higher (P < 0.05) than those in the RDS and non-RDS groups at 48 h, whereas the ACTG2 and TFF3 contents were significantly higher (P < 0.05) than the non-RDS group. A significant increase in the contents of IFABP (P ≤ 0.01), LFABP (P < 0.05), TFF3 (P < 0.05), ACTG2 (P < 0.05) at 48 h was detected in the RDS group only. In conclusion, the changes in the biomarkers support the suspicion of intestinal damage such as necrotising enterocolitis (NEC) after enteral feeding in premature calves with RDS. Intestinal damage biomarkers such as IFABP, LFABP, TFF3, and ACTG2 may be useful in the diagnosis of intestinal damage in premature calves. These results also indicate that the plasma concentrations of the intestinal biomarkers change in new born calves with their gestational age.