Toll-like receptors (TLRs) are essential for activation of the innate immune system in response to invading pathogens. In this paper, expression profiles of the turbot (Scophthalmus maximus) TLR22 gene (tbTLR22) were analyzed with RT-PCR and in situ hybridization. Then its expression patterns simulated with ligands or pathogens were investigated. Streptococcus iniae, Edwardsiella tarda, Hirame rhabdovirus virus (HIRRV), polyinosinic: polycytidylic acid (Poly I:C), peptidoglycan (PGN), or lipopolysaccharides (LPS) was injected to turbot; poly I:C, PGN, or LPS was added into cultured peripheral blood leukocytes (PBL); and then the tbTLR22 in liver, spleen, gill, kidney and cultured PBL was measured using Quantitative PCR. The recombinant protein of tbTLR22 (rp-tbTLR22) and its antibody were produced, then the reactions of antibody to tissues were detected by Western-blotting, and the binding of rp-tbTLR22 to all the stimulants was detected using ELISA. The results showed tbTLR22 expression was significantly up-regulated by PolyI: C, but no significant change in PGN and LPS groups; tbTLR22 significantly increased in liver and spleen after S. iniae infection with the maximum of 3.6 times and 3.3 times; in liver and kidney after E. tarda infection with the maximum of 3.4 times and 4.1 times; and then in gill and kidney after HIRRV infection by 4.8 and 4.1 times. Rp-tbTLR22 antibody could recognize the total protein from liver, kidney, gill and spleen at 40 kDa, 90 kDa and 120 kDa, respectively. The rp-tbTLR22 could bind to three ligands and pathogens in vitro. The expression and reaction data gave a clear recognization model of tbTLR22.