Immunoassays are nowadays being employed for rapid contaminant analysis in clinical, environmental, and agrochemical samples. A thorough characterization of the antibody‒antigen interaction can bring light to the immunoreagent selection process in order to develop sensitive and robust tests. Thus, determination of equilibrium and reaction rate constants is usually recommendable. However, this can be quite tricky for low molecular weight compounds, and competitive strategies are commonly followed to estimate apparent affinity values. In the present study, a collection of monoclonal antibodies to penthiopyrad was raised for the first time, and apparent equilibrium constants were assessed by the Langmuir model using three different competitive enzyme-linked immunosorbent assay formats. The obtained KD values from antibody-coated assays were quite close to the corresponding KD values calculated from surface plasmon resonance (SPR) evaluation. These studies were employed to select a pair of immunoreagents for immunoassay development. The KD value for penthiopyrad of the selected antibody obtained by SPR was 0.28 nM. The optimized direct assay showed an IC50 value for penthiopyrad of 0.42 nM (0.15 ng mL−1) in buffer. The limit of quantification for grape, must, and wine samples was 10 ng mL−1. An excellent correlation was found when immunochemical results were compared with those from LC-MS/MS. As an application case, it was determined that 58% of penthiopyrad was still found in wine after fermentation. Display omitted •The first monoclonal antibodies to penthiopyrad are herein reported.•Antibody-antigen reaction rate constants were determined by SPR.•Thermodynamic constants were estimated by different formats of competitive ELISA.•ELISA IC50 values approach KD values under limiting immunoreagent concentrations.•A direct immunoassay was validated to measure penthiopyrad in grapes and wine.