An accurate and simple preconcentration procedure was presented for cloud point extraction of Co2+, Ni2+ and Cu2+ from water, blood and urine samples. The procedure based on complexation of the metal ions with (E)-2-(2,4-dihydroxybenzylidene)-N-phenylhydrazine-1-carbothioamide (DHBPHC) and subsequent extraction into a surfactant-rich phase of Triton X-114. The optimized procedure (50 mL solution; pH 6; 1 × 10–4 mol L-1 of DHBPHC; 0.08% (v/v) Triton X-114) exhibits linear range up to 500 µg L-1 and limit of detection of 0.34, 0.94 and 0.83 µg L-1 for Co2+, Ni2+ and Cu2+, respectively. Most potentially interferants did not impact the extraction process. The procedure was applied for preconcentration and determination of Co2+, Ni2+ and Cu2+ in water, blood and urine by flame atomic absorption spectrometry.