Cortisol levels in bodily fluids represent a useful index for pituitary−adrenal function, and thus practical anti-cortisol antibodies are required. We have studied “antibody-breeding” approaches, which involve in vitro evolution of antibodies to improve their antigen-binding performances. Here, we produced an antibody fragment to measure serum cortisol levels with over 30-fold enhanced affinity after single mutagenesis and selection steps. A mouse anti-cortisol antibody, Ab-CS#3, with insufficient affinity for practical use, was chosen as the prototype antibody. A “wild-type” single-chain Fv fragment (wt-scFv; K _a, 3.4×10⁸ M⁻¹) was prepared by bacterial expression of a fusion gene combining the V _H and V _L genes for this antibody. Then, random point mutations were generated separately in V _H or V _L by error-prone PCR, and the resulting products were used to assemble scFv genes, which were displayed on filamentous phages. Repeated panning of the phage library identified a mutant scFv (scFv#m1-L10) with an over 30-fold enhanced affinity ( K _a 1.2×10¹⁰ M⁻¹). Three amino acid substitutions (Cys49Ser, Leu54Pro, and Ser63Gly) were observed in its V _L sequence. In a competitive enzyme-linked immunosorbent assay (ELISA), the mutant scFv generated dose–response curves with measuring range ca. 0.03–0.6 ng/assay cortisol, midpoint of which (0.15 ng/assay) was 7.3-fold lower than that of wt-scFv. Although cortisone, 11-deoxycortisol, and prednisolone showed considerable cross-reactivity, the mutant scFv should enable sensitive routine cortisol assays, except for measurement after metyrapone or high-dose of prednisolone administrations. Actually, cortisol levels of control sera obtained with the scFv-based ELISA were in the reference range.