A double T-wire time-resolved fluorescence rapid quantitative detection card of zearalenone with high sensitivity,good accuracy,fast and simple was successfully developed.By comparing the sensitivity of the number of detection lines(single T line,double T line)of ZEN detection card,the main advantage of double T line supplement is used to raise the sensitivity.The process of independent C-wire and non-independent C-wire system is compared.By using the non-independent C-wire process,the C value is negatively correlated with the T value to improve the sensitivity.The non-independent C-line process of sheep anti-mouse and automouse an-ti-mouse was screened,and the labeled antibody amount was further screened(5、10、20、30 μg/mL),and the la-beled antibody amount was 10 μg/mL,the titer was 25429,and the inhibition rate of 10~500 μg/kg was 33.57%~98.30%,and the sensitivity and titer were the best.HPLC and zellalenone(ZEN)time-resolved fluorescence rapid quantitative detection card were used to detect the samples of different substrates at the same time.The a-greement between the two was high,the accuracy was 90.89%to 119.33%,and the concentration was CV6.18%.The results show that this method has good stability,good shelf life and wide measuring range for the determina-tion of zalalenone in grain.