A sensitive magnetic particle enzyme-linked immunoassay (ELISA) was developed to analyze polybrominated diphenyl ethers (PBDEs) in water, milk, fish, and soil samples. The assay was rapid and can be used to analyze fifty samples in about 1h after sample cleanup. The assay has a limit of detection (LOD) below 0.1 ppb towards the following brominated diphenyl ether (BDE) congeners: BDE-47, BDE-99, BDE-28, BDE-100, and BDE-153, with the LOD approximately the same as GC-NCI-MS. The congeners most readily recognized in the ELISA were BDE-47 and BDE-99 with the cross-reactivities of BDE-28, BDE-100, and BDE-153 being less than 15% relative to BDE-47. As anticipated, the sensitivities are proportional to the similarities between the hapten structure and the BDE congener structure. Some oxygenated congeners with structural similarity to the hapten showed high to moderate cross-reactivities. Very low cross-reactivity was observed for other PBDEs or chlorinated environmental contaminants. The assay gave good recoveries of PBDEs from spiked water samples and a very small within and between day variance. Comparison with GC-NCI-MS demonstrated the ELISA method showed equivalent precision and sensitivity, with better recovery. The lower recovery of the GC-NCI-MS method could be caused by the use of an internal standard other than an isotopically substituted material that could not be used because of the fragmentation pattern observed by this method. The cleanup methods prior to ELISA were matrix dependent, no pretreatment was needed for environmental water samples, while fish, milk, and soil samples required various degrees of cleanup. Analysis of this wide variety of environmental samples by both ELISA and GC-MS demonstrated ELISA provides a timely and cost-effective method to screen for PBDEs in a variety of samples.