Toll-like receptors (TLRs) play important roles in fish innate immune and are involved in the defense process of bacteria invasion. In the present study, the full-length cDNA of TLR3 from the sea perch, Lateolabrax japonicus, was cloned and characterized. The full length of LjTLR3 cDNA was 3265 bp including an open reading frame of 2679 bp encoding a peptide of 922 amino acids. Tissues distribution analysis indicated that LjTLR3 showed a tissue-specific variation with high expression in spleen, head-kidney and liver. In order to investigate LjTLR3 functions against bacteria infection, the expression patterns of LjTLR3 after Vibrio harveyi and Streptococcus agalactiae challenge were detected by qRT-PCR, and the results showed that LjTLR3 was significant up-regulated after both bacteria stimulation in head-kidney, spleen and liver in a time-depended manner. Furthermore, the results by in situ hybridization experiments showed that positive signals of LjTLR3 mRNA in infected spleen and head-kidney were more numerous than that in the control group. In addition, intracellular localization revealed that LjTLR3 is distributed in the cytoplasm. In summary, these findings suggest that LjTLR3 was involved in the immune process under bacteria infection. This study would benefit to further clarify the roles of fish TLRs in the immune process and contribute to further study on enhancing disease resistance of L. japonicus.