Abstract: In order to achieve high sensitivity and rapid detection of folic acid, this study established a highly sensitive competitive enzyme-linked immunosorbent assay (cELISA) by preparing folic acid monoclonal antibody and optimizing the detection system. The results showed that when the concentration of coated antigen was 0.5 μg/mL, the dilution of antibody was 16000 times, and the dilution of secondary antibody was 10000 times, and the incubation time of primary antibody and secondary antibody and color development time were 15, 30 and 10 min respectively, the established cELISA method had half inhibition concentration of 1.12 ng/mL, and the limit of detection of 0.028 ng/mL. The method had good specificity and no cross-reaction with vitamin B₁, B₂, niacin and other B vitamins, the cross-reaction rates of dihydrofolic acid, tetrahydrofolic acid and pteric acid were 12.21%, 2.07% and 2.72%, respectively. The recoveries of milk and energy drink samples were 88.32%~94.12% and 88.00%~93.29%, respectively. The consistency rates of folic acid content in B complex vitamin tablets and infant milk powder were 102.01%~114.72% and 99.04%~111.62%, respectively, with relative standard deviation less than 5%. This study provides a sensitive, specific and accurate method for the rapid detection of folic acid content, which can improve the efficiency of field detection, and has a reference value for the rapid detection method of nutrient fortification content.