We employ photoluminescence (PL) spectroscopy on individual nanoscale aggregates of the conjugated polymer poly(3-hexylthiophene), P3HT, at room temperature (RT) and at low temperature (LT) (1.5 K), to unravel different levels of structural and electronic disorder within P3HT nanoparticles. The aggregates are prepared by self-assembly of the block copolymer P3HT-block-poly(ethylene glycol) (P3HT-b-PEG) into micelles, with the P3HT aggregates constituting the micelles’ core. Irrespective of temperature, we find from the intensity ratio between the 0–1 and 0–0 peaks in the PL spectra that the P3HT aggregates are of H-type nature, as expected from π-stacked conjugated thiophene backbones. Moreover, the distributions of the PL peak ratios demonstrate a large variation of disorder between micelles (inter-aggregate disorder) and within individual aggregates (intra-aggregate disorder). Upon cooling from RT to LT, the PL spectra red-shift by 550 cm^–1, and the energy of the (effective) carbon-bond stretch mode is reduced by 100 cm^–1. These spectral changes indicate that the P3HT backbone in the P3HT-b-PEG copolymer does not fully planarize before aggregation at RT and that upon cooling, partial planarization occurs. This intra-chain torsional disorder is ultimately responsible for the intra- and inter-aggregate disorder. These findings are supported by temperature-dependent absorption spectra on thin P3HT films. The interplay between intra-chain, intra-aggregate, and inter-aggregate disorder is key for the bulk photophysical properties of nanoparticles based on conjugated polymers, for example, in hierarchical (super-) structures. Ultimately, these properties determine the usefulness of such structures in hybrid organic–inorganic materials, for example, in (bio-)sensing and optoelectronics applications.