Self-sorting in thermoplastic elastomers was studied using bisurea-based thermoplastic elastomers (TPEs) which are known to form hard blocks via hierarchical aggregation of bisurea segments into ribbons and of ribbons into fibers. Self-sorting of different bisurea hard blocks in mixtures of polymers to give separate ribbons was established by studying exciplex formation between fluorescent pyrene and dimethylaniline (DMA) functionalized bisurea probes. If both probes had the same spacing between the bisurea units as the matrix hard segments, exciplex bands were observed in the fluorescence emission spectra, whereas exciplex intensities were strongly reduced when DMA and pyrene probes with different spacer lengths were incorporated in a mixed matrix that provided matching host segments for each guest separately. Probes with butylene spaced bisurea groups showed the highest degree of self-sorting. Self-sorting was further improved by annealing of the polymer films leading to a degree of self-sorting of more than 95%. Self-sorting at the fiber level of aggregation was studied by fluorescence resonance energy transfer (FRET) between naphthalene (donor) and pyrene (acceptor) bisurea guests. Large differences in FRET behavior for matching and nonmatching probes showed that significant self-sorting takes places between ribbons to give separate fibers.