Efficient expression of target protein is one of strategies for gene therapy or vaccine design. Many studies showed that codon optimization could enhance the expression of target proteins. In this paper, a target sequence of about 1.26 kb encoding the major capsid protein VP6 of grass carp reovirus (GCRV) and an optimized counterpart were synthesized and inserted into vectors for expressing VP6. The final constructs (named as pcDV6G and pcDV6YG) were transfected in Ctenopharyngodon idellus kidney (CIK) cells. The fluorescence analysis and the Western blot results showed that the gene fragment was transfected and expressed in CIK cells successfully. Although the qRT-PCR results showed no difference at the messenger RNA (mRNA) levels between the different versions of vp6 in the indicated stages, the enzyme-linked immunosorbent assay (ELISA) results showed that the protein level of VP6 expressed by pcDV6YG was higher than that by pcDV6G in the indicated hours. Taken together, these results suggest that the enhanced expression of GCRV VP6 in CIK cells by relative sequence optimization may be a good choice for making DNA vaccine against GCRV.