•We report an immunological method for detection of 2-substituted (1,3)-β-d-glucans.•The method can be used in the presence of other polysaccharides.•The method can quickly identify 2-substituted (1,3)-β-d-glucan producing bacteria.•The method can be used for the characterization of glycosyltransferases. Exopolysaccharides synthesized by lactic acid bacteria have prebiotic properties and contribute to the rheology and texture of fermented foods. Here, we have standardized an immunological method for the specific detection of 2-substituted (1,3)-β-d-glucans. The method allows direct detection and quantification of this exopolysaccharide in culture supernatants containing other mono- and poly-saccharides. Moreover, it allows specific detection of the biomolecules synthesized in vitro in enzymatic reactions. Thus, this method allows the fast identification of producing bacteria, as well as biochemical characterization of the glycosyltransferases responsible for their synthesis.