The fluorescence of a rhodamine X dye covalently linked to the 5' terminus of a 25-mers thymine oligodeoxynucleotide (dT25-ROX), adsorbed on the shell of thermoresponsive core-shell polymer particles, was used to probe the polarity, mobility, and distribution of the oligodeoxynucleotides (ODNs) in the shell. The particles have a glassy core of poly(methyl methacrylate) (PMMA) with a 67-nm radius, and a thermoresponsive shell of poly(N-isopropyl acrylamide) (PNIPAM) whose thickness changes from 42 nm at 11 ºC to 5 nm at 45 ºC. The variation in polarity of the shell with temperature was obtained both from the lifetimes and from the solvatochromic shifts of the dye and shows a sharp transition at the volume phase transition temperature (TVPT) of the PNIPAM shell. Förster resonance energy transfer (FRET) between dT25-ROX and a malachite green (MG)-labeled ODN (dT25-MG) was used to obtain the distribution of the ODNs in the thermoresponsive shell. Our results show that at 23 ºC (below TVPT) the ODNs are distributed inside the shell, sensing an environment similar to water. At this temperature, the PNIPAM shell is composed of hydrated chains with high mobility, as probed by the fluorescence anisotropy of dT25-ROX. By increasing the temperature above TVPT, the shell collapses and the chain mobility drastically slows down owing to the anchoring of the ODN to the dense shell of PNIPAM. Furthermore, FRET shows that the ODNs are absorbed on the 5-nm-thick collapsed shell but extend into the water. The polarity probed by the ROX averages the dyes distributed in the interior of the particle shell and in water, with 60 % of the dyes outside the particle shell (i.e., sensing pure water). Another indication that above the TVPT most of the ODNs are oriented with the dye toward the water phase is that the mobility of the dye covalently bound to the ODNs is identical in water and in the collapsed particle shell. The hybridization efficiency between an ODN supported in the particle shell (by adsorbing the ODN below TVPT and subsequently increasing the temperature above TVPT) and the complementary ODN in solution is identical to that of hybridization in water. This result opens good perspectives toward the use of the core-shell thermoresponsive nanoparticles as supports in DNA bioassays.