Toll-like receptor gene family is a class of conserved pattern recognition receptors, which plays an essential role in innate immunity providing efficient defense against invading microbial pathogens. Epinephelus akaara is one of the most important commercial marine fishes, and the main aquaculture industry in China is distributed along the coast of Fujian. The species is also considered a good model for studying immunity. Although TLRs have been extensively characterized in both invertebrates and vertebrates, a comprehensive analysis of TLRs in E. akaara is lacking. This research aims to study the systematic evolution, chromosome distribution, and expression regulation patterns of the Toll-like receptor genes in different tissues of E. akaara, based on the published genome and transcriptomic data of E. akaara, this study analyzed the phylogenesis, chromosome distribution, and expression regulation patterns of Toll-like receptor gene family in E. akaara tissues using bioinformatics methods including BLAST, phylogeny and synteny. The results showed that a total of 17 TLR genes were identified in E. akaara, which were divided into 5 subfamilies (TLR1, TLR3, TLR5, TLR7 and TLR11) and distributed on 11 of the 24 chromosomes. The 17 TLRs showed different expression patterns in different tissues. EaTLR1-2, EaTLR2-2, and EaTLR13-2 were mainly highly expressed in the spleen. EaTLR5M was highly expressed in the kidney, spleen, gills, and heart, but lowly expressed in other tissues. EaTLR5S was highly expressed in the kidney, liver, and spleen, but with low expression in other tissues. EaTLR18-1 and EaTLR18-2 were mainly expressed in the heart. EaTLR8 was highly expressed in all tissues except muscle and liver. EaTLR1-1, EaTLR2-1a, EaTLR2-1b, EaTLR3, EaTLR7, EaTLR9, EaTLR13-1, EaTLR21, and EaTLR22 were mainly highly expressed in the brain, spleen, kidney, and gill. In addition, EaTLR18-1 and EaTLR18-2 were both located on chromosome 9 with highly similar expression patterns. EaTLR2-1a and EaTLR2-1b were located on chromosome 20 and their expression patterns were also highly similar. In addition, EaTLR5M and EaTLR5S, which had high LRR domain similarity, were located on chromosome 14, and had high similarity in expression levels in different tissues. On the contrary, EaTLR7 and EaTLR8, which were located on chromosome 18, and EaTLR2-2 and EaTLR3, which were located on chromosome 4, had different expression patterns. These suggested that copies of TLR genes in the same chromosome may have similar expression patterns or functions. This work provided reference data for studying the evolution of the Toll-like receptor system in fish, and laid a foundation for further research on the function of Toll-like receptor genes in E. akaara.