Intestinal fatty acid-binding protein (I-FABP), a low molecular mass (approximately 15 kDa) cytoplasmic protein, is specifically located in epithelial cells of small bowel mucosal layer. This protein is rapidly released into the circulation after injury and/or destruction of these cells due to poor mesenteric blood flow and necrosis. Therefore, it can be used as a potential diagnostic biomarker for small bowel disease. In the present study, we have succeeded in developing a sandwich enzyme-linked immunosorbent assay (ELISA) system for quantification of human I-FABP. The range of sandwich ELISA system was 0.1-50 ng/mL of I-FABP in serum, and showed excellent quantitative characteristics such as reproducibility, dilution linearity, and recovery. No cross-reactivities were detected with other types of FABPs. As measured with this ELISA system, the serum I-FABP concentration was 1.1 ± 0.9 ng/mL in 61 healthy individuals, indicating that the reference value was below 2.0 ng/mL regardless of gender and age. Furthermore, mild abdominal pain or diarrhea before blood sampling did not affect I-FABP levels. Thus, this ELISA system could be used to accurately quantify human I-FABP concentrations in serum samples. These results suggest that it could be used as a new biomarker for the diagnosis of small bowel disease.