Fluorescent probes, coumarin 153 (C153) and octadecylrhodamine B (ORB), were used to study the self-assembly in water of poly(N-decylacrylamide)-block-poly(N,N-diethylacrylamide), (PDcA11-block-PDEA295; Mn= 40 300 g mol−1; Mw/Mn= 1.01). From the variation of both the fluorescence intensity and the solvatochromic shifts of C153 with polymer concentration, the critical micelle concentration (CMC) was determined as 1.8 ± 0.1 μM. On the other hand, steady-state anisotropy measurements showed the presence of premicellar aggregates below the CMC. Time-resolved fluorescence anisotropy evidenced that ORB is located in the premicellar aggregates and the micelle core, while C153 is partitioned between the aggregates and the water phase. The micelle core contains both semicrystalline and amorphous regions. In the semicrystalline regions the probes cannot rotate, while in the amorphous regions the rotational correlation times correlate well with the hydrodynamic volume of the probes. The amorphous region of the micelle core is relatively fluid, reflecting the large free-volume accessible to the probes.