The antigen‐binding domain of an antibody specific for the herbicide atrazine was cloned from hybridoma 4063–21–1 and expressed as a single‐chain antibody (scAb) in the vector pPM1‐His. The observed heavy and light chain gene sequences were consistent with those in subgroup VH I (B) and VK TV, as defined by the Kabat database of sequences. Subsequent expression in Escherichia coli strain XL‐1 Blue produced up to 0.3 μg of functional single‐chain antibody, from periplasmic protein preparations, per millilitre of culture. The scAb was purified as a monomer by immobilized metal chelate affinity chromatography via a hexa‐histidine tail or as a dimer by antibody affinity chromatography. The functionality and specificity of the recombinant antibody was confirmed by binding to an atrazine‐bovine serum albumin conjugate and free atrazine and simazine in a competition ELISA. This is the first example of the production and purification of a recombinant antibody that retains antigen binding for a triazine herbicide.