Sorghum mosaic virus (SrMV) is a virus that causes mosaic symptom on sugarcane worldwide. In recent years, SrMV is becoming the most predominant pathogen for sugarcane mosaic disease (SMD) in Guangxi province, the major sugarcane-growing region in China. To facilitate the detection of SrMV, high throughput and sensitive serology-based detection techniques were developed and compared with RT-PCR assay. A high titer polyclonal antibody against bacterial-expressed recombinant coat protein of SrMV was first generated. The specificity and sensitivity of the resultant antibodies were evaluated by Western blotting, indirect-ELISA and Dot-ELISA. SrMV could be detected in crude sap at dilution 1:100 by Dot-ELISA, 1:1000 by indirect-ELISA, and at 1:100,000 by IC-RT-PCR. Meanwhile, a non-reactive result was found in SrMV-free extracts. In a randomly collected field sample pool, 60% of the samples showed SrMV-positive by IC-RT-PCR, similar to that of RT-PCR assay, compared with 46% by indirect-ELISA or 48% by Dot-ELISA. Besides, IC-RT-PCR, Dot-ELISA and indirect-ELISA methods achieved an agreement with RT-PCR procedure of 90, 86 and 78% for the detection of SrMV, respectively. Taken altogether, our findings indicate that IC-RT-PCR is a simple, sensitive and reliable method for SrMV detection, which holds a great potential for SMD control, quarantine programs and the diagnosis of SrMV in asymptomatic infected plant samples.