A simple, low cost, fast and environmentally safe cloud point extraction procedure (CPE) for separation and preconcentration of iron (Fe(III)) in different environmental water samples prior to its spectrophotometric determination has been developed. The CPE method is based on the reaction of iron(III) with N,N'-bis(salicylidene)ethylenediamine (Salen) as a reagent yielding a complex at pH 4.0, respectively which extracted in a mixed micelle system consists of Triton X-114 (non-ionic surfactant) and cethyltrimethyl ammonium bromide (CTAB) at λmax 612 nm. The main parameters affecting the CPE efficiency, such as the pH of the solution, Salen concentration, surfactant concentration, incubation time and temperature were investigated and optimized. Under the optimum conditions, the calibration curve was linear in the range of 5.0-400 µg L−1 with R2 ≤ 0.9996. The detection limit was 1.5 µg L−1 with preconcentration factor was 50. The intra-day and inter-day precision (as the relative standard deviation, RSD%) was 1.75 and 2.0%, respectively. The interference of various cations and anions has been studied. The proposed CPE procedure was successfully applied for the determination of trace amount of Fe(III) in various water samples with high efficiency and satisfactory results.