Functionalized biosensing surfaces were developed for chemiluminescent immunoassay of pesticides. Two approaches to construct functionalized surfaces were tested: (i) pesticide is immobilized to the surface and interacts with a labeled antibody; (ii) antibody is immobilized and interacts with a labeled pesticide. As labels alkaline phosphatase and peroxidase were used with their corresponding substrates CSPD and luminol, respectively. Light produced by chemiluminescent substrate was detected by a thermoelectrically cooled CCD camera or a photomultiplier. The best detection limit 0.00001 ng/ml was obtained using antibodies immobilized to dextran-enhanced surface. Completely renewable surface was obtained using reversible lectin-monosaccharide interaction, one surface was used for 200 analyses without any loss of binding capacity. Most favorable stability and cost per analysis was achieved with molecularly imprinted polymer (MIP) instead of antibody. The functionalized biosensing surfaces were prepared to detect 2,4-dichlorophenoxyacetic (2,4-D) acid as a model pesticide. The developed concepts are, however, generally applicable to other pesticides and to other optical formats, e.g. optical fiber.