In this study, various factors of ELISA for detection of sulfamethazine residues were explored, the coating antigen was diluted to 1:400, the best coating condition was at 4℃ overnight, the working concentration of HRP-IgG enzyme conjugate was 1:7 000. The pre-incubation time and incubation time was 30 min and 120 min, respectively, the substrate solution working time was 20 min. Two moL· L-1 H2SO4 was used to stop the reaction and checked. A standard curve of direct competitive ELISA had been established to detect the sulfamethazine residues in milk. The detection limit of this method was 1.97 ng·mL-1. The mean concentration of sulfamethazine required to inhibit 30% antibody was 7.1 ng·mL-1. The linear range of the detection was 5-200 ng·mL-1. The recovery ratio was between 73.20% and 91.16%. The CV% of within array and between arrays was less than 10%.