Abstract. The Layer-by-Layer (LbL) method using polyelectrolytes is a typical strategy for preparing functional polymeric multi-layered films. Besides the electrostatic force between positively and negatively charged polyelectrolytes, the hybridization force between complementary DNA strands can be also used for building the DNA LbL films. Herein, we report the thermal responsible DNA LbL film that is designed to release specific DNA oligomers (10 bases) at an elevated temperature. Because the temperature and the ionic strength (IS) of the medium affect the hydrogen bonds in the double stranded DNA, the structural stability of the film against temperature and IS was investigated. The loading and release of the releasable oligomers were repeatedly performed on the DNA LbL film.