Objective To establish a duplex real-time polymerase chain reaction (PCR) method for simultaneous detection of the Ruvettus pretiosus and Lepidocybium flavobrunneum. Methods The specific primers and probes were designed base on cytochrome C oxidase subunitⅠ gene of Ruvettus pretiosus and Lepidocybium flavobrunneum, respectively. The perfromance of method was assessed, including sensitivity, specitivity and repeatability. The method was applied to detect the Ruvettus pretiosus and Lepidocybium flavobrunneum from fish products. Results The result indicated that duplex real-time PCR method was specific to Ruvettus pretiosus and Lepidocybium flavobrunneum. The method showed good linear relationship between Ct value and sample copies in 8.4×10⁵-8.4×10⁰ copies/μl, and the linear regression equation were y=-3.18x+41.0(R²=0.998 8) and y=-3.37x+44.5(R²=0.998 6) for Ruvettus pretiosus and Lepidocybium flavobrunneum, respectively. The limit of quantification (LOQ) was 16.8 copies. The relative standard deviation (RSD) of this duplex real-time PCR was 0.29%-0.84%, the RSD was 0.29%-0.84% and 0.29%-0.62% for Ruvettus pretiosus and Lepidocybium flavobrunneum, respectively. The Ruvettus pretiosus were identified using this method from 50 codfish products. Conclusion This duplex real-time PCR was a sensitive, sepecific and stable method. It would be useful for dete-ction of the Ruvettus pretiosus and Lepidocybium flavobrunneum.