A simple strategy to achieve molecular recognition in water is to make the polymers self-organized into nanospheres which could incorporate the functional groups containing hydrogen bonding sites into hydrophobiclipophilic regions. A novel amphiphilic polymer, Poly(polyoxyethylene-600)-oxy-5-(6-(1-thymine)hexyl)) isophthaloyl (PPETHI), has been synthesized. The PPETHI polymer could self-organize into nanospheres in dilute aqueous solution, which were 150–300 nm in diameter as estimated by SEM. The isophthaloyl with side hexyl thymine of the polymer selforganized into hydrophobic regions and the PEG surrounded it. Molecular recognition between thymine in PPETHI polymer and adenine substrate has been studied by FT-IR. The FT-IR studies demonstrate that C4=O of thymine has recognized with N-H of adenine through complementary nucleobases. It shows that the typical characteristic band at 3,352 cm?1 of N-H stretching vibration of adenine shifted to 3,373 cm?1 and the band at 1,685 cm?1 of C4=O of thymine shifted to 1,680 cm?1. To confirm the formation of hydrogen bonds, the N-H band at 3,373 cm?1 and C4=O band at 1,680 cm?1 have retrieved to 3,312 cm?1 and 1,685 cm?1 respectively upon heating to 115 °C or higher by means of variable temperature FT-IR. The formations of hydrogenbonds between thymine in the polymer and adenine substrate in nanospheres were confirmed. It could enhance their interaction and loading capacity.