Abstract: Carbofuran was once a highly toxic carbamate pesticide widely used in the prevention and control of pests in the agricultural field. In recent years, the problem of excessive residues of carbofuran in agricultural products has been reported, posing a great threat to human beings, mammals, and the environment. The maximum permitted levels of carbofuran in food have been established under different legislations worldwide. Therefore, it is necessary to strengthen supervision and rapid detection of carbofuran residues in food. Due to its high solubility and high yield, nanobody has been paid more attention in recent years. To further improve their sensitivity and stability to meet the demands of actual detection, the Nb5 was selected for in vitro evolution research. The structure models of Nb5 were constructed based on AlphaFold and verified by Ramachandran Plot, ERRAT, and Verify 3D. The key amino acid sites of Nb5/carbofuran were determined by LeadIT software and alanine scanning. The result showed that the carbofuran molecule was inserted into the pocket composed of CDR2, FR3, and CDR3. Nb5 and carbofuran were driven by hydrogen bonds and hydrophobic forces. Based on the confirmed mutation sites (Arg56, Trp58, Thr101, Val102, Ala103, Asp104, Cys105), a phage display saturation mutation library was established to screen mutants, and their performance was characterized. The IC₅₀ of H10 was 17.6 ng/mL, the sensitivity was twice compared with that of the wild type Nb5, and the affinity constant was 1.54 × 10⁶ L/mol. This study provides an effective idea for the affinity maturation of nanobodies against small molecule drugs in vitro.