A cloud point extraction (CPE) process was described for the separation and spectrophotometric analysis of Fe(III) in different water samples and blood as chelating agent. The complexation reaction between Fe(III) and Zincon was elaborated at pH 5.0 using acetate buffer and the complex was quantitatively recovered in a mixed micelle system composed of cethyltri methyl ammonium bromide (CTAB) and Triton X-114. This reaction was carried out at room temperature in the presence of 0.05 mol L-1 Na2SO4 as salting-out electrolyte. The linearity was up to 1000 µg L-1. The preconcentration factor was 50. The precision (as relative standard deviation) and the limit of detection were 2.5% and 3.1 µg L-1, respectively. The proposed procedure was used for the spectrophotometric detection of Fe(III) in water and blood samples and the data were statistically comparable to those achieved using ICP-OES technique.