The application of Fourier transform infrared (FTIR) spectroscopy to the study of poly(ethylene oxide)poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) block copolymer is shown to give valuable information concerning the formation of a hydrophobic microenvironment in aqueous solutions. FTIR spectra of Pluronic P103 in H2O and D2O solutions were measured as a function of temperature. The temperature dependence of FTIR spectral data indicates that the methyl groups in PPO blocks of the polymer are experiencing a progressively less polar environment with an increase of temperature, and the strength of hydrogen bonds formed by the ether oxygen in PO groups with water is reduced by heat. The dehydration of PO segments favors the formation of a hydrophobic microenvironment in aqueous solutions. At higher temperatures, the appearance of the band of the dehydrated methyl groups in the FTIR spectra of aqueous Pluronic P103 solutions is related directly to the aggregation of PEO-PPO-PEO block copolymer and the formation of a hydrophobic microenvironment., The proportion of the dehydrated methyl groups increases with an increase of temperature; Pluronic P103 would form a micelle with a denser PPO core in aqueous solutions at higher temperatures.