A ELISA kit for detection of Sulfamethoxazole (SMZ-Kit)was developed with generating hybridoma lines that secrete SMZ monoclonal antibodies (SMZmAb) and the method of blocking ELISA,its traits were identified. The calibration curve of SMZ-Kit was typical sigmoid curve fitted to the four parameter logistic equation with the linear detection of 1.0 to 128.0μg/ L (R² = 0.9901),the sensitivity of 0.73μg/ L,the IC₅₀ of 11.5μg/ L and the detection limit of 1.0μg/L.The recoveries of SMZ spiked in feed were 83.9 % and in milk were 83.3 %.The precision and accuracy of the assay as determined by inter-assay and intra-assay coefficient variation were below 15 %.The SMZ - Kit had 2.88 % cross-reactivity towards Sulfadiazine (SD) and little or no cross-reactivity towards other Sulfonamides.The validity of SMZ-Kit in 4℃was above six months.