Adulterating meat products with several species, including non-halal species, is often found in commercial products. Therefore, non-halal ingredients are a major source of concern for Muslims. Multiplex polymerase chain reaction (PCR) with multiple primers can detect contamination of meat components from non-halal species in a single reaction process, making it more effective and efficient. Multiplex PCR is a PCR technique that uses multiple primers and DNA samples in one reaction to amplify multiple target regions. In this study, a pair of species-specific primers encoding the Cytochrome c oxidase subunit I (CO1) gene were designed to amplify bovine DNA, tested for specificity, and applied in multiplex PCR technique together with D-loop primers for pigs, Cyt-b for rats, and 12S rRNA for dogs. The CO1 primers, along with D-loop primers for porcine, Cyt-b primers for rats, and 12S rRNA primers for dogs, can be used to detect specific bovine DNA with a size of 279 bp and sequence similarity of 96%.