Golden pompano (Trachinotus ovatus) is an important economically fish species. In this study, with an aim to identify reliable reference genes for quantitative real-time PCR (qRT-PCR) in golden pompano, we evaluated the expression stability of eight housekeeping genes in the presence and absence of poly I:C stimulation in eight tissues. The PCR data was analyzed by geNorm and NormFinder algorithms. The results showed that the expression of all the examined genes exhibited tissue-dependent variations. When under normal physiological condition, geNorm and NormFinder identified B2M and 18S as suitable genes. When studying gene expression under conditions of poly I:C stimulation, the selection of the internal controls should be selected on a tissue basis. At 12 h stimulation, geNorm ranked Actin/UBCE, Actin/B2M, UBCE/B2M, Actin/UBCE, RPL13/B2M, UBCE/GAPDH, B2M/RPL13, and UBCE/B2M, respectively, as the most stably expressed genes in liver, spleen, kidney, gill, intestine, heart, muscle, and brain. Comparable ranking orders were produced by NormFinder. Similar results were obtained at 48 h stimulation. Taken together, these results indicate that B2M and 18S are the most stable gene across tissue types under normal physiological conditions. However, during poly I:C stimulation, no single gene or single pair of genes in the examined set of housekeeping genes can serve as a universal reference across all tissue types. If one gene is preferred, B2M, B2M, UBCE, Actin, B2M/RPL13, B2M, B2M, and RPL13 may be used in spleen, kidney, liver, gill, intestine, brain, muscle, and heart of golden pompano, respectively.