Intestinal permeability is directly related to intestinal homeostasis and individual health. At present, there is a lack of quantitative evaluation methods and reagents for intestinal permeability of fish. Serum intestinal fatty acid binding protein (I-FABP) is an important serum biological index for quantitative evaluation of intestinal permeability. In order to establish a quantitative detection method of serum I-FABP concentration in crucian carp (Carassius auratus), the I-FABP gene of crucian carp was cloned, expressed and purified in prokaryote. The rabbit and mouse polyclonal antibodies were prepared by immunizing New Zealand white rabbit and mouse with the recombinant protein, and their titers were 1:80 000. The rabbit polyclonal antibody was used as the coating protein, and the mouse polyclonal antibody labeled with HRP was used as the enzyme labeled antibody. By optimizing the reaction conditions, a double antibody sandwich ELISA (DAS-ELISA) method based on rI-FABP was established. The linear regression equation of the standard curve was y=0.003 5x+0.036 7, the fitting degree was high, R² was 0.999 1, and the cut-off value was 0.090 6. The intra- and inter-assay coefficient of variation was 1.17%-5.50% and 0.16%-4.78% indicating the good repeatability of established DAS-ELISA. Compared with a commercial kit, the coincidence rate among 54 samples was 100%. The DAS-ELISA detection method constructed in this study can be used for the quantitative evaluation of the intestinal permeability of crucian carp. In view of the conservatism of fish I-FABP, this method may be used for quantitative evaluation of intestinal permeability of other fish.