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The recombinant single-chain trispecific antibody against three toxins including Okadaic acid (OA), saxitoxin (STX) and tetrodotoxin (TTX) was developed. Three single-chain variable fragment (ScFv) genes were linked via linkers (Gly4-Ser)3 to construct fusion ScFv gene OST-ScFv through splicing by overlapping extension. Fusion gene OST-ScFv was 2 256 bp, encoding 752 amino acids, and was inserted into expression vector pET-22b. Recombinant plasmid OST-ScFv-22b was expressed at 37°C and at 25°C by induction with β-D-1-thiogalactopyranoside. The expressed proteins were identified by SDS-PAGE and purified by urea dissolving, gradient dialyzing and ultrafiltrating. Molecular weight of expressed protein was about 79.000 kDa. Competitive indirect enzyme linked immunosorbent assay indicated that the single-chain trispecific antibody which had different combining activity with three toxins was obtained. This work confirmed that multi-specific ScFv antibody could be obtained via linking expression of ScFvs and might also have an application in synchronization detection of toxins.