This paper presents a method based on the cloud point extraction for the separation and preconcentration of Se (IV) and Se (VI) in environmental water samples as well as total selenium in animal blood and tissue samples. 3,3'-Diaminobenzidine (DAB) is a selective and sensitive reagent and is known to form an intense yellow compound piazselenol with selenium (IV). When a system consisting of sample, DAB and surfactant Triton X-114 is warmed above the cloud point of the surfactant, it was seen that the DAB-Se (IV) complex gets extracted into the surfactant rich phase while the Se (VI) remains in the aqueous phase. Se (VI) in the sample was reduced to Se (IV) by microwave heating of solution in 4 mol L-1 HCl and total Se was estimated by carrying out the CPE. The quantification of selenium was carried out using ETAAS. The analytical parameters for the quantitative cloud point extraction of the Se-DAB complex were investigated and optimized. The proposed procedure was validated by applying it to the determination of the content of Se in Certified Reference Material BND 701-02. (NPL, India). The detection limit of selenium in environmental water samples was 0.0025 mu g L-1 with an enrichment factor of 100. The relative standard deviation (RSD) for ten replicate measurements of 5 mu g L-1 was 3.6%. The proposed method was successfully applied to the determination of selenium (IV), (VI) in environmental water samples and determination of total selenium in human blood, SRM-IAEA-A-13 animal blood and SRM-IAEA-407 fish tissue. (C) 2009 Elsevier B.V. All rights reserved.