Toll-like receptors (TLRs) are central players in the innate immune system in response to a wide range of pathogen infection. Among various TLRs, TLR4 plays a key role in recognition of bacterial lipopolysaccharides (LPS). In the present study, we identified and characterized a novel TLR4 homologue (maTLR4b) in blunt snout bream (Megalobrama amblycephala) which was significantly distinct from previously reported M. amblycephala TLR4 (tentatively named maTLR4a). The results showed that the complete cDNA sequence of maTLR4b was 3261 bp with an open reading frame encoding a polypeptide of 820 amino acids, and that its genomic sequence was 3793 bp, which had 3 exons. Structurally, the deduced maTLR4b protein showed a typical TLR domain architecture, including a signal peptide, eight leucine-rich repeats (LRRs) in the extracellular region, a transmembrane domain, and a Toll-Interleukin 1 receptor (TIR) domain in the cytoplasmic region. Phylogenetic analysis revealed that all TLR4s from teleost fish formed a monophyletic clade. Both maTLR4a and maTLR4b were divided into two distinct branches, and showed the highest level of similarity with the grass carp TLR4.2 and TLR4.4 homologue, respectively. MaTLR4b was constitutively expressed in all healthy tissues tested although at different levels. After LPS stimulation, the expression levels were significantly up-regulated in spleen, and peaked at 4 h between maTLR4a and maTLR4b, but with a distinct and complementary expression patterns. Taken together, these results suggested that maTLR4b is indeed a functional homologue of TLR4 in other species, which may play vital role in innate immune.