The invention discloses a multiple RT-PCR detection primer group for detecting five kinds of sugarcane viruses simultaneously. The multiple RT-PCR detection primer group for detecting five kinds of sugarcane viruses simultaneously comprises six pairs of PCR primers which aim at five sugarcane viruses such as SCBV, SSV, SCMV, ScYLV and SrMV and one sugarcane reference gene GPADH correspondingly; the six pairs of PCR primers have base sequences as shown in a sequence table SEQ ID NO.1 to SEQ ID NO.12 correspondingly. Therefore, primer concentration and annealing temperature which influence multiple RT-PCR amplification are optimized by a multiple RT-PCR system and a corresponding RT-PCR detection method is established; and by the method, the five viruses for infecting the sugarcane can be detected simultaneously from sugarcane tissues rapidly, accurately, simply and conveniently, and important significance in screening healthy seedling mother plants, monitoring healthy seedling breeding,breeding for disease resistance and predicting field disease epidemic is achieved. In order to facilitate application of the multiple RT-PCR detection primer group, a corresponding kit is designed and assembled, so that popularization and promotion in grass roots are facilitated.