In order to detect rapidly and efficiently the content of carminic acid (CA) in food, the artificial antigen of CA was prepared by hydroxydiimidazole method; A high-titer polyclonal antibody (Pab) against CA was obtained through immunizing New Zealand white rabbits to obtain antiserum (against CA) for further separation and purification by ammonium sulfate precipitation. The titer of the antibody was tested by checkerboard titration, and the effects of factors such as solution pH, ionic strength, and organic solvent were investigated to optimize the conditions of the enzyme-linked immunosorbent assay (ELISA) reaction. Under the optimal conditions, an indirect competitive ELISA inhibition curve for CA was established. Ultraviolet (UV) spectroscopy analysis showed that both the immunogenic antigen (CA-BSA) and coating antigen (CA-OVA) were successfully conjugated with carrier protein. The titer of the Anti-CA polyclonal antibody obtained after separation and purification was 1:16000; The optimal concentration of the detection antigen was 1 μg/mL, the optimum buffer for ELISA reaction was the phosphate buffer at pH 7.4 containing 50 mmol/L Na+ but no methanol. The linear range of the indirect competitive ELISA standard curve was 7.8~1150 ng/mL, with the limit of detection (LOD) as 1 ng/mL, and the sensitivity IC50 was 95.2 ng/mL. This study has laid the foundation for the development of a rapid CA rapid test kit for the determination of CA in food.