A simple and efficient cloud point extraction-spectrofluorimetric method for the determination of thiamine in human urine is proposed. The procedure is based on the oxidation of thiamine with ferricyanide to form thiochrome, its extraction to Triton X-114 micelles and spectrofluorimetric determination. The variables affecting oxidation of thiamine, extraction and phase separation were studied and optimized. Under the experimental conditions used, the calibration graphs were linear over the range 2.5-1000 ng $mL^{-1}$. The limit of detection was 0.78 ng $mL^{-1}$ of thiamine and the relative standard deviation for 5 replicate determinations of thiamine at 400 ng $mL^{-1}$ concentration level was 2.42%. Average recoveries between 93-107% were obtained for spiked samples. The proposed method was applied to the determination of thiamine in human urine.