An enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative analysis of alkylphenol polyethoxylates (APnEOs) and their biodegradation products. To generate a specific monoclonal antibody (mAb) for the ELISA, hybridoma cells were produced by the fusion of mouse myeloma cells and spleen cells from mice immunized with nonylphenol polyethoxylate (NPnEO) derivatives coupled to bovine serum albumin. The developed ELISA showed the detection limits of 16 and 30 microg/L NP10EO when 10% and 60% (v/v) methanol solutions were used as assay diluent. The mAb was shown to be specific to APnEOs and their metabolites, such as short-ethoxy-chain APnEOs and alkylphenoxy carboxylic acids, except for nonylphenol. Moreover, no response was observed with non-APnEO surfactants as well as other compounds structurally similar to APnEOs. The percentage river water recoveries of 85-118% were obtained for 10 microg/L NP10EO fortification after preconcentration by C18 solid-phase extraction. The ELISA was also validated by comparing it with high-performance liquid chromatography for the analysis of APnEOs and their metabolites in river samples; the correlation coefficient between the values obtained by these assays was 0.96.