A recombinant bispecific single-chain diabody (scDb), recognizing fluoroquinolones (FQs) and sulfonamides (SAs), was successfully constructed with two single-chain variable fragment antibodies (scFvs). The scDb gene was cloned into the expression vector pJB33, and 6xHis-tagged scDb was expressed as soluble bodies in Escherichia coli RV308 host, then purified by one step affinity chromatography of immobilized metal ion affinity chromatography (IMAC). SDS-PAGE and Western blotting analysis of the purified scDb indicated that the prepared scDb was successfully expressed as a similar to 60 kDa and the final purity of the scDb protein was up to 95% with yields of approximately 6 mg/L of bacterial culture. The scDb was further characterized by indirect competitive enzyme linked immunosorbent assay (icELISA), showing that the affinity and specificity of scDb were fully retained from the two parental scFvs, capable of simultaneously binding FQs and SAs. The 50% inhibition concentration (IC50) values of the optimized immunoassay were 0.45 ng mL(-1) for FQs and 0.75 ng mL(-1) for SAs, respectively. The scDb exhibited high affinity to 20 FQs and 14 SAs. Taken together, these findings suggested that the prepared scDb could be used to develop future novel immunoassay for simultaneous determination of 20 FQs and 14 SAs. (C) 2014 Elsevier Inc. All rights reserved.