Abstract: To establish a multiplex fluorescent quantitative PCR for the detection of tobacco mosaic virus (TMV), cucumber mosaic virus (CMV) and potato virus Y (PVY), specific primers and probes were designed based on the conserved CP gene sequences of TMV, CMV and PVY, respectively. And the reaction system was optimized, subsequently the sensitivity, specificity, repeatability and accuracy were evaluated. Results showed that the detection limit for TMV, CMV and PVY was 2.60×10¹, 1.25×10¹, 2.33×10¹copies/μL, respectively. The detection sensitivity was 10 times higher than the conventional PCR method. No cross-reaction was found with tobacco etch virus (TEV), tobacco vein banding mosaic virus (TVBMV) and tomato spotted wilt virus (TSWV). The coefficient of variation between intra-assay and inter-assay were both under 1.5%. By using the triple fluorescent quantitative PCR, 24 samples were detected and the positive rate of TMV, CMV and PVY was 75%, 100%, 41.67%, respectively, which were consistent with the single-PCR. In conclusion, a specific, stable and accurate triple fluorescent quantitative PCR was established and could be used for diagnosis and epidemiological investigation of TMV, CMV and PVY.