A recombinant single chain variable-fragment (scFv) antibody against paeoniflorin (PF) was produced using the hybridoma cell line C31B9. Variable regions of heavy (V (H)) and light (V (L)) chain antibody genes were directly cloned from cDNA resources of hybridoma C31B9 and assembled using splicing by overlap extension (SOE)-PCR using a (Gly (4)Ser) (3) linker DNA. The constructed scFv genes were cloned into pET28a vectors for the generation of recombinant proteins in Escherichia coli. Most of the recombinant proteins were expressed in inclusion bodies. The yield of refolded and purified scFv was 1.89 mg per 100 mL of cell culture. The recombinant scFv displayed cross-reactivity as its mother monoclonal antibody (MAb) C31B9. Therefore, the newly expressed scFv protein was applied to quantitative ELISA to determine the total paeoniflorin (PF) and albiflorin (Alb) concentrations in peony root samples. Using PF as a standard compound, the full linear range of the assay was extended from 0.78 to 25 microg/mL. The results obtained by ELISA employing both the recombinant scFv and the original MAbC31B9 showed a reasonably good agreement with each other.