Objective: A multiplex fluorescence PCR method based on TaqMan probe was developed for simultaneous detection of soybean and wheat allergens in food. Methods: TaqMan probe dual PCR was designed and established based on wheat gliadin gene and soybean lectin gene, respectively. The soybean Lectin gene was detected with FAM marker, and the wheat gliadin gene was detected with HEX marker. Moreover, the eukaryotic 18S rRNA was used as internal reference gene to ensure the effectiveness of the detection system. Results: The multiplex fluorescence PCR method for detection of soybean and wheat allergens created in this study had no cross-reaction to ingredients other organisms showing strong specificity. The detection limit of soybean and wheat mixture was up to 0.01%(mass ratio), and the detection sensitivity was high. Conclusion: The established TaqMan probe real-time multiplex detection system for soybean and wheat allergens has the characteristics of good specificity and high sensitivity, which can be used for simultaneous and rapid detection of soybean and wheat allergens in food.