In order to establish a multiplex polymerase chain reaction (PCR) method for simultaneous detection of five tetracycline resistance genes including tetA, tetD, tetG, tetS and tetX in poultry bacteria, the concentrations of primers and Taq DNA polymerase, as well as the annealing temperature used in the multiplex PCR system were optimized, then the sensitivity and specificity were examined, and finally, the method was applied for detecting of tetracycline-resistant bacteria isolated from poultry to verify its applicability. The results showed that, the optimized system for the multiplex PCR reaction was: 5 μL of mixed DNA template, 1 μL of Taq enzyme, 0.5 μL of forward and reverse primers for tetA and tetX, and 1.0 μL for tetG, tetD and tetS. 4 μL dNTP mixtures, 4 μL MgCl₂, 5 μL 10×PCR buffer, and finally filled to 50 μL with double distilled water. The reaction procedures inculding: predenaturation at 95 ℃ for 5 min, denaturation at 95 ℃ for 30 s, annealing at 55 ℃ for 30 s, extension at 72 ℃ for 1 min, 35 cycles, finally extension 72 ℃ for 10 min. The sensitivity of the method was 10² CFU/mL, moreover, a broad spectrum of poultry bacteria could be detected for the five tetracycline resistance genes by the method. Compared with the traditional single PCR method, the multiplex PCR technique is rapid, efficient and widely applicable.