A recombinant single-chain variable fragment (scFv) antibody, specific for the organophosphate pesticide parathion-ethyl, was produced from genetic material obtained from a hybridoma cell line secreting an anti-parathion monoclonal antibody (Mab). The scFv and parent Mab showed similar, but not identical, characteristics in a microtitration plate enzyme-linked immunosorbent assay (ELISA). Limits of detection for the Mab and scFv-based ELISAs of 1.6 and 2.3 ng/well, respectively, were observed. Cross-reactions of other structurally similar organophosphates were similar in the two assays; highest cross-reactions were observed with parathion-methyl and fenitrothion (both giving 6% and 4% values for the Mab-and scFv-based assays, respectively). Performances of the assays in the presence of methanolic extracts of rice and orange peel were compared; the scFv demonstrated similar stability to the Mab at methanol concentrations up to 5% (v/v). At higher concentrations (above 10%, v/v) performance of both assays deteriorated, though the Mab-based assay was less affected than the scFv-based assay. ScFvs promise to be as useful as Mabs for the analysis of food samples by ELISA.