This experiment established a relative quantitative detection method for bovine derived components in milk powder based on Real time PCR. The specificity, sensitivity, and stability of cattle-specific primers and probes were tested. By simulating mixed samples of cow’s milk powder and horse’s milk powder with different concentrations, linear fitting was performed based on the functional relationship of their △Ct values, and a standard curve was drawn to establish a relative quantitative detection of bovine derived components in milk powder. The results showed that the minimum detection limit of this method was 0.000 01 mg/mL. The recovery rate was 91.11%~119.2%, with an inter group coefficient of variation of ≤0.58% and an intra group coefficient of variation of ≤1.44%. This method is suitable for detecting the adulteration of bovine derived components and content in milk powder in terms of specificity and stability.