A single-chain Fv fragment (scFv) against estradiol-17β (E2) was generated to begin the construction of a library of various mutated anti-steroid antibodies with an improved affinity and/or specificity. A hybridoma clone secreting a specific anti-E2 antibody (Ab#E4-4) was established by the cell fusion using splenocytes from a mouse immunized with an immunogenic E2-carrier conjugate. DNA fragments encoding the variable heavy and light domains (VH and VL) of the Ab#E4-4 were cloned and combined to give the scFv gene fragment encoding the sequence 5′-VH-(GGGGS)3-VL-3′. Compared to the Ab#E4-4 Fab fragment, soluble scFv (scFv#E4-4) protein showed a similar affinity to E2 (Ka = 8.6 × 107 M−1) and a similar cross-reaction profile. To further study the fundamentals for creating a comprehensive library of mutated scFvs, the scFvVH and VL genes were amplified using error-prone PCR conditions and the frequency and pattern of incorporated mutations were investigated. For this, regular Taq polymerase was used in the presence of unequal concentrations of dNTPs. At 1.0 mM MnCl2, the error frequency reached to 8.5% and 11% for the VH and VL respectively, although a significant transition/transversion bias was observed. ScFv#E4-4 and the mutated polyclonal scFvs were then displayed on filamentous phage under various packaging conditions. Cultivation of the transformed bacteria was more suitable at 25 °C than at higher temperatures for the packaging of scFv-bearing phagemid particles. Based on these experimental conditions, an scFv-displaying phage library, each scFv member in which has mutated complementarity-determining region (CDR) H2, H3, L1, and L3, was constructed. A soluble scFv clone (scFv#m1-e7) with a mutated amino acid (I → V) in CDR L1, isolated from this library, showed threefold higher affinity (Ka = 2.6 × 108 M−1) than that of scFv#4-4.