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In order to find fraudulent species substitution in meat products, a highly sensitive and rapid assay for meat species identification and quantification is urgently needed. In this study, species-specific primers and probes were designed from the mitochondrial cytb (cytochrome b) fragment for identification and quantification of bovine ingredient in commercial meat products. Bovine samples and non-bovine ones were used to identify the specificity, sensitivity, and applicability of established assay. Results showed that the primers and probes were highly specific for bovine ingredient in meat products. The absolute detection limit of the real-time PCR method was 0.025 ng DNA, and the relative detection limit was 0.002% (w/w) of positive samples. The quantitative real-time PCR assay was validated on simulated meat samples and high in the precision and accuracy. In order to demonstrate the applicability and reliability of the proposed assay in practical products, the 22 commercial meat products including salted, jerkies, and meatball were used. The results indicated the established method has a good stability in detection of bovine ingredient in real food. The established method in this study showed specificity and sensitivity in identification and quantification of beef meat in processed meat products.