We propose a new method that double-stranded DNA molecules can be stretched and immobilized on the clean glass substrate by using a lipid monolayer at the air-water interface. This method is based on the substrate lifting of Langmuir-Blodgett method. We observed fluorescence images of polyion complex films with a scanning near-field optical microscope (SNOM). As a result, straight fluorescent lines aligned parallel to the lifting direction were observed and it was considered that isolated single DNA molecules were extended to align on the substrate. This method is applied to various DNA molecules.