Expression of four reference genes of grass carp, including beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA (18S) and elongation factor-1 alpha (EF1 alpha), was studied in tissues of normal individuals and bacteria-infected individuals. EF1 alpha had the most stable expressions followed by 18S rRNA then GAPDH; ACTB had the least stability. After being infected with bacteria, the grass carp showed minimal changes in expression levels of EF1 alpha in the liver and head kidney, while ACTB had the most stable expressions in spleen but the least stable in liver. EF1 alpha is thus the optimal reference gene in quantitative real-time PCR analysis to quantitate the expression levels of target genes in tissues of grass carp.