Botulism is caused by botulinum neurotoxin (BoNT), the most poisonous substance known. Potential use of BoNT as a biothreat agent has made development of sensitive assays for toxin detection and potent antitoxin for treatment of intoxication a high priority. To improve detection and treatment of botulism, molecular evolution and yeast display were used to increase the affinity of two neutralizing single chain Fv (scFv) antibodies binding BoNT serotype A (BoNT/A). Selection of yeast displayed scFv libraries was performed using methods to select for both increased association rate constant (k_on) and decreased dissociation rate constants (k_off). A single cycle of error prone mutagenesis increased the affinity of the 3D12 scFv 45-fold from a K_D of 9.43×10⁻¹⁰M to a K_D of 2.1×10⁻¹¹M. Affinity of the HuC25 scFv was increased 37-fold from 8.44×10⁻¹⁰M to 2.26×10⁻¹¹M using libraries constructed by both random and site directed mutagenesis. scFv variable region genes were used to construct IgG for use in detection assays and in vivo neutralization studies. While IgG had the same relative increases in affinity as scFv, (35-fold and 81-fold, respectively, for 3D12 and HuC25) higher solution equilibrium binding constants were observed for the IgG, with the 3D12 K_D increasing from 6.07×10⁻¹¹M to 1.71×10⁻¹²M and the HuC25 K_D increasing from 4.51×10⁻¹¹M to 5.54×10⁻¹³M. Affinity increased due to both an increase in k_on, as well as slowing of k_off. Higher affinity antibodies had increased sensitivity, allowing detection of BoNT/A at concentrations as low as 1×10⁻¹³M. The antibodies will also allow testing of the role of affinity in in vivo toxin neutralization and could lead to the generation of more potent antitoxin.