In forensic toxicology, immunoassays for drug screening are widely used because of the simple test procedures and instantaneous outcome of results. However, commercial immunoassay products are available for only a limited number of drugs. Preparation of antidrug antibodies is a crucial, but time-consuming in creating an immunoassay system. In this study, we focused on the application of a single-chain variable fragment (scFv) antibody for drug screening and developed a fluvoxamine (FLV) detection system for indirect competitive enzyme-linked immunosorbent assay (icELISA) using the scFv against FLV. To clarify the influence of domain order on the scFv binding activities, we prepared two kinds of scFv with different domain orders (HL, V-H-linker-V-L, and LH, V-L-linker-V-H) and examined their kinetic parameters against FLV. The scFvs showed sufficient FLV binding activities (K (D) = 3.8 and 7.6 nM), and the HL scFv was slightly more favorable for FLV binding than the LH scFv. The developed icELISA using the HL scFv could detect FLV in the range of 10-200 ng/mL, and the scFv has no cross-reactivity below 100 mu M except for chlorpromazine and imipramine. We also quantified the plasma FLV concentrations in forensic autopsy cases, and the results showed that this method could be applied effectively for FLV quantification without the need for extraction steps. Although recombinant antibodies against small molecule drugs for immunoassays have not yet been commonly used, we can predict that they could be a powerful tool to screen drugs in the near future, because of their advantages.