Abstract: Objective: To establish a multiplex real-time PCR assay capable of simultaneously and rapidly identifying bovine, chicken, duck and porcine components in beef products and thus to counteract economically motivated adulteration with cheaper meats. Methods: Species-specific primers and TaqMan probes targeting the mitochondrial cytochrome b (Cytb) gene were designed and screened for cattle, chicken, duck and pig. A single-tube reaction system was constructed and systematically validated for sensitivity, specificity, linearity and adulteration simulation. Results: The assay exclusively amplified DNA from cattle, chicken, duck and pig, with no cross-reactivity against 6 non-target species. The method simultaneously detected four animal-derived DNA with 10⁻⁴ ng/μL sensitivity and a 0.01% (w/w) detection limit. Screening of 100 retail beef samples revealed 24 adulterated products, predominantly containing duck meat and/or pork. All results were fully concordant with those obtained by the national standard method. Conclusion: The developed multiplex real-time PCR protocol is rapid, cost-effective and high-throughput, significantly enhancing the capability to detect meat adulteration and providing robust technical support for food-safety regulation and consumer protection.