To establish a multiplex real-time fluorescence RT-PCR detection method of norovirus GI, GII and hepatitis A virus in strawberry, and apply to actual sample.Methods After pretreatment of strawberry samples, viruses were concentrated, extracted and purified for virus RNA. The samples were determined by real-time fluorescence RT-PCR and multiplex real-time fluorescence RT-PCR. The specificity, sensitivity, and practical applications of the methods were also analyzed. Results The nucleic acid extraction method could effectively remove the inhibitory factors. The methods showed high specificity with no cross amplifications of other virus. The detection limit of multiplex real-time fluorescence PCR for norovirus GI, GII and hepatitis in strawberry was 56.2 RT-PCR₅₀/20 g, 31.6 RT-PCR₅₀/20 g and 31.4 CCID₅₀/20 g, respectively. According to the test results of 50 inspection samples, the results were all negative.Conclusion The established system of nucleic acid extraction and real-time fluorescence RT-PCR detection method is suitable for the simultaneous detection of norovirus GI, GII and hepatitis A virus in strawberry.