Abstract: Taking catalase activity as an indicator, we studied on the method suitable for on-site rapid detection of grain mildew. The results showed that the coefficient of variation of the detected value of a detection fungal suspension prepared by high-speed stirring method was the smallest, and the detection time was 1/20 to 1/10 that of the conventional methods such as oscillatory washing method. The detection solution preparation conditions had significant effects on the results. The grain epidermis damage rate could be reduced by preparing the detection solution under the conditions of adding water to the grain samples without soaking, stirring for 40 s while rotating at 2 300 r/min, thereby obtaining stable detection results. The established method was applied for mildew detection of different grain species and varieties, and the results showed that the correlation coefficient between rapid detection value of enzyme activity and the conventional detection results of fungal carrying capacity reached 0.99 or more. The results of enzyme activity and fungal carrying capacity were 527 U/100 g and 1.6×10⁵ cfu/g, respectively when the content of mildewed grain was 2%. The correlation coefficients of enzyme activity and fungal carrying capacity for different proportion of mildewed grain were 0.99 and 0.97, respectively. Therefore, the established rapid detection method of enzyme activity can better reflect the mildew status of grain and meet the needs of on-site grain rapid detection.