A TaqMan real-time polymerase chain reaction (PCR) assay was developed for the simultaneous detection of chicken-, duck-, swine- and bovine-derived ingredients in quick-frozen food. Specific primers and TaqMan probes were designed and synthesized using the transforming growth factor beta-3 (TGFB3) gene of chicken, the prion protein (PRNP) gene of swine and the growth hormone (GH) gene of duck and bovine as target genes. The PCR reaction systems and conditions were optimized. The developed real-time PCR assay demonstrated good specificity, in which specific amplifications were observed for chicken, duck, swine and bovine. The assay also showed high sensitivity with limits of detection for chicken-, duck-, swine- and bovine-derived ingredients of 10.0, 1.0, 10.0 and 10.0 pg/μL, respectively. The PCR assay could detect the four kinds of animal-derived ingredient in 34 commercial quick-frozen foods. Further analysis revealed that the results of the PCR assay for 15 quick-frozen foods were inconsistent with the ingredients on the label. The real-time PCR assay developed in this study has good specificity and sensitivity, and it is simple to operate and suitable for the rapid detection of animal-derived ingredients in quick-frozen foods.