In this study, adulteration of bovine milk in fresh and thermally processed non-bovine milk was detected by SYBR Green and TaqMan real-time polymerase chain reaction (real-time PCR), and the effect of different heat treatment methods on the adulteration detection was explored aiming to meet the requirements for the detection of adulteration of different commercial non-bovine milk products. The results showed that the designed bovine-specific primers allowed specific amplification of DNA in bovine milk without cross-reactivity with DNA from non-target animals. The minimum detection limits of SYBR Green and TaqMan real-time PCR for bovine milk DNA were 1 and 10 pg, respectively, and both methods were capable of detecting at least 0.1% (m/m) bovine milk adulteration in non-bovine milk mixtures. The results of nine repeated experiments over three days confirmed high reproducibility of the methods. In addition, using the real-time PCR methods, bovine milk was detected in 7 out of 20 commercial non-bovine milk products, indicating high resolution and great practical application value of these methods.