Current immunoassays for herbicide detection are usually based on polyclonal or monoclonal antibodies (MAbs) raised in animals. The mammalian expression system allows the procurement of specific and highly sensitive antibodies, avoiding animal immunization. In this study, S-metolachlor-specific IgG vectors bearing either Thosea asigna virus 2A or internal ribosome entry site (S-T2A or S-IRES) and single-chain variable fragment (scFv) vectors were designed and expressed. The recombinant antibodies (RAbs) were characterized by indirect competitive enzyme-linked immunosorbent assays (icELISA). The results showed that full-length RAbs exhibited significantly better performance than scFv, and both bicistronic vectors expressed antibodies of correct size, while RAb S-T2A elicited a higher yield than RAb S-IRES. Further analyses showed that RAb S-T2A and RAb S-IRES exhibited comparable reactivities and specificities to the parental MAb, with IC50 values of 3.44, 3.89 and 3.37 ng/mL, respectively. Finally, MAb- and RAb-based icELISAs were established for the determination of S-metolachlor in environmental waters. The recoveries were in the range of 73.0–128.1%, and the coefficients of variation were mostly below 10%. This article describes the production of RAbs for S-metolachlor from mammalian cells for the first time and paves the way to develop RAb-based immunoassays for monitoring herbicide residues in the environment. Display omitted •A mammalian expression system was established to produce RAbs specific for S-metolachlor.•The antibody performance of scFv, and two full-length antibodies bearing T2A or IRES were compared.•The prepared full-length RAbs showed high affinity that preserved the parental MAb properties.•The sensitive and reliable icELISAs were developed based on full-length RAbs.•The developed icELISAs were applied for determination of S-metolachlor in environmental waters.