The full length cDNA of toll-like receptor 3 were cloned from Carassius auratus gibelio gill by means of reverse transcription polymerase chain reaction （RT-PCR）and rapid amplification of cDNA ends（RACE）. Sequence analysis revealed a 3170 bp cDNA containing 181 bp 5 ’-untranslated region，283 bp 3 ’-untranslated region and 2706 bp open reading frame（ORF）which encoded 901 amino acids . The deduced amino acid sequence of Carassius auratus gibelio had high similarity with that of Carassiu auratus(goldfish)，Danio rerio，Ictalurus punctatus，Oncorhynchus mykiss and Takifugu rubripes. The similarities were 92%, 88%, 79%, 75% and 72%，respectively. The full-length TLR3 of Carassius auratus gibelio contained a signal peptide in the first 21 amino acid residues, 15 leucine-rich regions (LRR) from 50th to 695th amino acid residue, a transmembrane region (TM) from 704th to 726th residue, a Toll/IL-1R homologous region (TIR) from 754th to 897th residue. Alignment with amino acids sequence of TIR from Carassius auratus gibelio, Homo sapiens and Danio rerio indicated, Phe728、Leu738、Tyr756 and Arg736 of Carassius auratus gibelio played an important role both in the receptor signaling and intracellular localization. The three-dimensional structure of Carassius auratus gibelio TLR3 ectodomain (TLR3-ECD) showed horseshoe-shaped structure and had high similarity with that of human TLR3-ECD. Carassius auratus gibelio TLR3-ECD possessed 1 disulfide bond at N-terminal and 2 at C-terminal. The extensive β-sheet on the molecular’s concave surface formed a platform several modifications, including insertions in the LRRs and 14 N-linked glycosylation sites.