Fluorescence spectra and dynamics of pyrene doped in segmented poly(urethane urea) (SPUU, copolymer of toluene-2, 4-diisocyanate (TDI) and poly(propylene oxide) (PPO)) films are studied by means of time-resolved total internal reflection (TIR) fluorescence spectroscopy. The differences in the environmental polarity and aggregates of pyrene between the surface layer (ca. 52 nm) and bulk layer (>0.68 μm) in the SPUU films are elucidated. The present results on the fluorescence characteristics and dynamics strongly indicate that pyrene molecules in the surface layer of the film are located in the vicinity of polar TDI segments and those in the bulk layer are incorporated near less polar PPO segments. Although the local concentration of pyrene in the surface layer is higher than that in the bulk layer, the excimer formation is less feasible in the surface layer than in the bulk layer, due to the formation of nonemissive ground-state pyrene dimer in the surface layer.