Accessibility of water-soluble fluorescence quencher, thallium nitrate (T1(+)), to pyrene (Py) sites of the Py-labeled random copolymer of sodium 2-(acrylamido)-2-methylpropanesulfonate and 50 mol % N-dodecylmethacrylamide (PAMPS-DodM-Py) was investigated with and without addition of the unlabeled random copolymer of N-isopropylacrylamide and 1.5 mol % N-dodecylacrylamide (PNIPAM-DodA) in aqueous solutions. The results revealed that the accessibility of T1(+) ions to the Py sites of PAMPS-DodM-Py was decreased by adding PNIPAM-DodA to PAMPS-DodM-Py, anticipating that more stable hydrophobic microdomain was formed due to the interaction between PAMPS-DodM-Py and PNIPAM-DodA.