We focused on the influence of temperature variations on macromolecule partitioning between unconfined and nanoconfined areas in nanoslits using single molecule measurements. We evaluated the number of fluorescent poly(ethylene glycol) (PEG) molecules confined in glass nanoslits in several configurations by fluorescence correlation spectroscopy (FCS) and recorded a decreasing trend in molecule partitioning within the confined environment when increasing the temperature. The trend is size-dependent, demonstrating the manipulation and size separation of a bimodal solution of uncharged macromolecules confined in nanochannels. These findings can have an impact on molecule manipulation and concentration, so far achieved mainly adopting external fields operating on charged molecules.