A rapid method, immunoaffinity column-ultra-high performance liquid chromatography, was established for determining zearalenone (ZEN) in grain. The retention volume of ZEN was 0.47 mL, the detection limit 5 pg. The linear detection ranges of ZEN was 10~500 pg with correlation coefficients (R2) of 0.999 9. Recovery rates in grains (wheat, corn) spiked with ZEN was in the range of 85%～102%, 83%～112%, respectively, with the relative standard deviation of 2.7%~6.9%. The method with analytical time less than 45 min was suitable for the determination of ZEN in the raw grain, with the advantages of simplicity, rapidness, sensitivity, good reproducibility, and less solvent.