The present invention discloses a DBN based separation method of a mixture of dual-tracer single-acquisition PET signals labelled with the same isotope. It predicts the two separate PET signals by establishing a complex mapping relationship between the dynamic mixed concentration distribution of the same isotope-labeled dual-tracer pairs and the two single radiotracer concentration images. Based on the compartment models and the Monte Carlo simulation, the present invention selects three sets of the same radionuclide-labeled tracer pairs as the objects and simulates the entire PET process from injection to scanning to generate enough training sets and testing sets. When inputting the testing sets into the constructed universal deep belief network trained by the training sets, the prediction results show that the two individual PET signals can been reconstructed well, which verifies the effectiveness of using the deep belief network to separate the dual-tracer PET signals labelled with the same isotope.