A flow immunoassay method for the measurement of 2,4-dichlorophenoxyacetic acid (2,4-D) was developed. The competitive fluorescence immunoassay relies on the use of antibody- or antigen-coated poly(methyl methacrylate) particles (98 µm diameter) as a renewable solid phase. The assay exhibits a dynamic range of 0.1–100 µg l^–1 using a monoclonal antibody or alternatively 10 µg l^–1 to 10 mg l^–1 using commercially available antiserum. The assay is demonstrated in buffered saline solution as well as in aquatic environmental media. The relative errors for the environmental matrices were similar to those for the buffer control. The precision of concentration values calculated at 1 mg l^–1 (for the assay using antiserum) were ±0.28, ±0.27 and ±0.43 mg l^–1 for the buffer, well water and river water matrices, respectively. The method shows cross-reactivity with compounds of closely related structure but little cross-reactivity with compounds dissimilar in structure to 2,4-D. The proposed automated competitive immunoassay method is rapid (between 7 and 15 min per assay), simple and potentially portable.