[Background] Colibacillosis and salmonellosis are the most common bacterial diseases in poultry, causing economically devastating to poultry industries. Salmonella and avian pathogenic E. coli (APEC) are important zoonotic pathogens, also with potential threat to human health. Thus, it is necessary to strengthen the rapid detection and differential diagnosis of these bacterial diseases. [Objective] We developed multiplex PCR to efficiently detect APEC, Salmonella enteritidis, Salmonella typhimurium, Salmonella gallinarum and Salmonella pullorum. [Methods] The special genes of APEC, Salmonella enteritidis, Salmonella typhimurium, Salmonella gallinarum and Salmonella pullorum were screened and selected for primers designing. A multiplex PCR method was developed by optimization of conditions. Then, the sensitivity and usage for detection of clinical isolates were determined. [Results] A multiplex PCR was established for accurately and effectively detection of APEC, Salmonella enteritidis, Salmonella typhimurium, Salmonella gallinarum and Salmonella pullorum. The sensitivity of the multiplex PCR was 103 bacterial colony forming units (CFUs) and 100 pg genomic DNA. The results of multiplex PCR for detection of clinical isolates agreed well with those of traditional serological assays. [Conclusion] Multiplex PCR developed in this study could efficiently detect APEC, Salmonella enteritidis, Salmonella typhimurium, Salmonella gallinarum and Salmonella pullorum.