Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) has become one of the most commonly used techniques for RNA expression. To obtain more reliable results with biological significance, it requires data normalization using an appropriate internal control gene. Here, we cloned partial sequence of elongation factor 1a (EF1a) gene from grass carp (Ctenopharyngodon idella). The stabilities of four commonly used internal control genes encoding 18S rRNA, b-actin, EF1a, and lyceraldehyde-3-phosphate dehydrogenase (GAPDH) were integratedly assessed using the geNorm, NormFinder and BestKeeper programs. Integrative analyses of qRT-PCR data indicated that the stability ranking of the examined genes was 18S rRNA> EF1a> GAPDH> b-actin in gill, head kidney, heart, intestine, liver, muscle, skin, spleen, and trunk kidney tissues in untreated grass carp. hen the same calculations were done in spleen tissue at different time points post grass carp reovirus (GCRV) infection, the gene ranking was 18S rRNA> b-actin> GAPDH> EF1a. The rank ordering of expression stability was EF1a>b-actin > 18S rRNA>GAPDH in C. idella kidney (CIK) cell culture stimulated by poly(I:C). The recommended ranking was EF1a> GAPDH> b-actin> 18S rRNA in CIK cells infected by GCRV. The results indicated that 18S rRNA was the best invariant internal control gene in individual level in grass carp, EF1a was the most suitable in CIK cell culture stimulated by poly(I:C) or infected by GCRV. As an assay, EF1a was employed to examine the changes of Toll-like receptor 3 (TLR3) and melanoma differentiation associated gene 5 (MDA5) after virus infection in CIK cells. These data laid the foundation for more precise results in qRT-PCR studies of gene expression in grass carp.