Monoclonal antibody (MAb) against trans-resveratrol (t-RSV) was obtained from hybridoma clones constructed from splenocytes of BALB/c mice immunized with carrier proteins (bovine serum albumin [BSA] and ovalbumin [OVA]) coupled with synthetic hapten mimicking t-RSV structure. The t-RSV-BSA derivate was more efficient at induction of the immune response than t-RSV-OVA. However, the use of t-RSV-OVA was advantageous during selection of hybridoma clones constructed from splenocytes of t-RSV-BSA-immunized mice. Pre-incubation of immune serum with free t-RSV inhibited the binding of antibody to t-RSV-BSA conjugate, suggesting the specific nature of antibody binding to t-RSV. Splenocytes obtained from the mouse immunized with t-RSV-BSA were used for the hybridoma construction. Expansion of the primary clones, their subsequent screening, and subcloning narrowed our search to allowed isolation of two IgG1a-producing hybridomas designated as 2H9 and 1B1. According to an indirect ELISA assay, the resulting MAb 2H9 had little or no cross-reactivity to cis-RSV. No recognition of trans-RSV-3-O-glucuronide and trans-RSV-3-sulfate was detected. The newly generated MAb against t-RSV may provide a highly valuable and cost-effective tool for the analytical assay for t-RSV in different biological and agricultural specimens.