In this study, a rapid, specific, and low-cost duplex-detection technique of pork and beef was developed by a real-time polimerase chain reaction assay based on fluorescence. Deoxyribonucleic acid was extracted from variable mixtures of pork and beef in sausage and industrial products to develop the duplex assay using the GIDAGEN((R)) Multi-Fast DNA Isolation Kit. Identification of genomes was accomplished in the same tube by their distinctive melting peak, which was 87.5 degrees C for pork and 80.5 degrees C for beef, respectively. The detection limit of the method was 0.01 ng/mu L deoxyribonucleic acidor 0.001% target pork and beef in sausage. The results showed that the intercalating dye based duplex real-time polimerase chain reaction is a potentially sensitive, reliable, and practical assay for the detection of meat species adulterated with beef and pork.