Soybean is susceptible to viral diseases which are often present as mixed infections. The individual simplex RT-PCR methods used for the identification of multiple viruses are more tedious and time-consuming than the corresponding multiplex RT-PCR. This study used soybean mosaic virus (SMV), bean common mosaic virus (BCMV) and cucumber mosaic virus (CMV)-infected leaf samples from southern China as the test materials to evaluate a multiplex RT-PCR assay developed for the simultaneous detection of these viruses. The parameters optimised included the annealing temperature, extension time, number of cycles, and primer type and concentration. The specific fragments sizes obtained by the multiplex RT-PCR were 550 bp (SMV), 288 bp (BCMV) and 99 bp (CMV). The assay was tested using infected soybean samples obtained from farmers’ fields in Sichuan Province, China. The multiplex RT-PCR assay had high sensitivity, was rapid and simple, and could be used for the diagnosis of soybean infected with various combinations of these viruses in the field.