The invention discloses a real-time polymerase chain reaction (PCR) detection primer, a kit and a detection method for detecting puffer fish ingredients. The puffer fish gene detection primer and probes with the sequences of SEQ ID NO. 1-3 are designed according to the specific gene sequence of puffer fish, and the real-time fluorescent PCR method is adopted to qualitatively detect the puffer fish ingredients of food. The developed real-time fluorescent PCR qualitative detection method for detecting the puffer fish ingredients in food has importance significance in improving the detection efficiency and sensitivity of the puffer fish ingredients in food, lowering the detection cost, detecting related fake products on the market, monitoring food safety and improving a detection technology, and has wide development prospect.