Being a foodborne virus, Rotavirus was often carried by shellfish. At the present, RT-PCR was the most effective method of Rotavirus detection in shellfish, but its sensitivity was low because of low levels of virus contamination and PCR inhibitors in shellfish. So contaminated shellfish experimentally in laboratory, and imitated the natural environment to concentrate Rotavirus, then detected by the developed single-tube seminested RT-PCR. Compared with ELISA and the conventional RT-PCR, the detection sensitivity is improved by 1000 times and 10 times respectively. In addition, the outer and inner contamination is reduced dramatically and the detection time is decreased from 6h to 4.5h. When the whole shellfish and only the digestive tissues of shellfish serve as samples, the detection ratio and sensitivity are more higher when sample is the latter.