Time-resolved fluorescence spectroscopy is used to observe subtleties in supramolecular structure during the self-assembly of polymers in solution. Lifetime distribution analysis of the fluorescence decay kinetics of the solvent-sensitive fluorescent probe 1-anilino-8-naphthalene sulfonic acid associated with the di-block copolymer poly(2-vinylpyridine)41–poly(ethylene oxide)204 (P2VP-PEO) as it self-assembles enabled identification of three microdomains, distinguishable on the basis of micropolarity. These microdomains can be assigned to different supramolecular substructures: the micelle corona (high polarity), the micelle core and the P2VP globule (both low polarity), and the core–corona interface and the globule–PEO junction (both intermediate polarity). Changes in the relative population distributions of these sub-structures as a function of P2VP-PEO pinpoint the onset of micellization corresponding to the critical micelle concentration (CMC) of the copolymer, but indicate significant variation in supramolecular structure, including micelle formation, well below the CMC. This suggests that supramolecular self-assembly in polymeric systems has characteristics of a second order phase transition.