In order to rapidly determine aflatoxin B1 (AFB1), zearalenone (ZEN) and deoxynivalenol (DON) in maize, a AFB1-ZEN-DON three-in-one combined detection method has been established. This quantitative immunofluorescence assay based on superconductor coating was used to detect multiple maize samples. Combined with the recovery rate, stability, detection limit, precision and other indexes, the applicability of the method were evaluated and compared with the results of liquid chromatography. The results showed that the AFB1-ZEN-DON three-in-one combined detection method had the good linearity in the range of 0.5~30 µg/mL for AFB1, 5~200 µg/kg for ZEN and 100~2 500 µg/mL for DON, respectively. The relative deviation of AFB1, ZEN and DON detected by this method was –2.84%~11.91%, –3.43%~18.12% and 1.83%~12.91%, respectively, which were within the allowable deviation of 20%, 15% and 23%. The detection limit of AFB1 was 0.41 μg/kg and the corresponding limit of quantification was 1.12 μg/kg. The limit of detection of ZEN was 3.22 μg/kg and the corresponding limit of quantitation was 9.15 μg/kg. The detection limit of DON was 35.42 μg/kg and the corresponding limit of quantitation was 100.84 μg/kg. Compared with the results of liquid chromatography, the superconductor coated immunofluorescence test strip method is an effective, practical, rapid and quantitative analysis method within the allowable deviation range, suggesting that it could meet the requirements of simultaneous detection of AFB1, ZEN and DON in maize.