Japanese meagre (Argyrosomus japonicus) is well-known for its high nutritional and economic value. In recent years, with the rapid expansion and intensification of aquaculture, Japanese meagre culture has suffered serious economic losses due to infectious diseases. Toll-like receptors (TLRs) can recognize highly conserved structural motif pathogen-associated microbial patterns (PAMPs) and danger-associated molecular patterns (DAMPs), playing a key role in resisting diseases by initiating host innate immune responses. Here, a total of 12 members of the TLR gene family (TLR1, TLR2, TLR2b, TLR3, TLR5M, TLR5S, TLR7, TLR9, TLR13, TLR14, TLR22, and TLR23) were identified in the genome of Japanese meagre. The TLR genes in Japanese meagre were distributed on 10 of 24 chromosomes. The evolutionary analyses showed that 12 different TLR genes were divided into different subfamilies, including the TLR1, TLR3, TLR5, TLR7, and TLR11 subfamilies. The conserved motifs were compared with three other teleost species. The results revealed that the conserved motifs in the same subfamilies were highly consistent. The identity and similarity of the TLR-TIR domain of Japanese meagre demonstrated that the regulatory mechanisms of each of the subfamilies were similar. qRT-PCR assays showed different expression patterns of TLRs in the liver, spleen, head kidney, trunk kidney, brain, and heart of Japanese meagre, but the expression levels in immune tissues were higher. Double-stranded RNA virus (dsRNA) is a common etiological agent of diseases in aquaculture species. Therefore, a synthetic double-stranded RNA called poly (I:C) was used as an injection to understand the immune system function and disease control of Japanese meagre. Following poly (I:C) stimulation, seven of twelve ArgTLR genes, including ArgTLR2, ArgTLR2b, ArgTLR3, ArgTLR7, ArgTLR13, ArgTLR22 and ArgTLR23, were significantly upregulated. In summary, our results not only play a guiding role in disease prevention and control and vaccine development in the future, but also provide basic data for future study of the TLR gene family of Japanese meagre.