Large yellow croaker (Larimichthys crocea), the most developing fish of China, suffering from both biotic and abiotic stressors. A genome-wide study was performed for the first time to investigate the roles of Toll-like receptor genes in large yellow croaker (LcTLRs) working in hypoxia response and Aeromonas hydrophila infection. 12 TLR genes were identified and annotated, and phylogenetic tree, analysis of structure and motifs demonstrated that TLRs were highly conserved. To further investigate the LcTLRs under environmental stress, expression pattern analysis of hypoxic response and Aeromonas hydrophila infection revealed that there were nine and six members of LcTLRs showing significant differential expression, indicating that they were concerned in hypoxia stress and disease responses. Meanwhile, their expression levels were validated utilizing qPCR. Taken together, a greater appreciation and understanding of TLRs function in the reaction to biotic and abiotic stress would ultimately lead to more efficiently environmental adaptation in large yellow croaker.