Plant-based meat alternatives (PMAs), as an emerging food category gaining increasing popularity, face potential food safety risks and ethical concerns for vegetarians due to the illegal adulteration of animal-derived components. To address these challenges and enhance regulatory oversight, the development of a rapid, sensitive, and highly specific detection method is essential. In this study, five DNA extraction methods were evaluated and optimized to identify the most effective approach for PMA products. The optimal conditions were determined to be 60 mmol/L NaCl, 10 mmol/L Tris HCl, and a centrifugation speed of 12,000× g. Additionally, specific primers targeting four common animal-derived adulterants, namely pork, chicken, duck, and beef, were designed and screened for targeted amplification. To establish a rapid and visually interpretable detection system, the recombinant polymerase amplification conditions were optimized. The final protocol used 0.4 µmol/L primer and isothermal amplification at 39 °C for 25 min, with the incorporation of SYBR Green I dye enabling the rapid and specific visualization of animal-derived DNA. This optimized method is characterized by its simplicity, sensitivity (capable of detecting beef-derived components as low as 0.0514% w/w), and rapidity, significantly reducing detection time and providing a reliable tool for the identification of animal-derived adulteration in PMA products.