Abstract: OBJECTIVE To optimize the multiplex PCR system of aflatoxin producing fungi and to determine the optimal PCR system. METHODS Multiple PCR primers were designed based on the sequences of ver-1, verB and ITS, which were the main regulatory genes in aflatoxin production, DNA template, Mg²⁺concentration, primer dosage, dNTPs dosage, annealing temperature and other factors were investigated by single factor and orthogonal test. RESULTS The optimal multiplex PCR system was as follows:50 mL system contained primer(5 µmol·L^-1) 3 µL, dNTPs(2.5 mmol·L^-1) 5 µL, Mg²⁺(2.5 mmol·L^-1) 4 µL, DNA concentration 10 ng·µL^-1, annealing temperature 56℃. CONCLUSION The established system can be used for the identification of aflatoxin producing fungi by multiplex PCR and has certain significance for the source identification of aflatoxin producing fungi.