In a hybridized microchannel made of poly(dimethylsiloxane) (PDMS) and glass, nonuniform electroosmotic flow (EOF) was created when an electric field was applied. PDMS and glass pieces were bonded at room temperature after oxidization with oxygen plasma. After oxidization, the PDMS surface has a higher zeta (zeta) potential than that of glass, which results in faster EOF at that surface. When the channel has a large aspect ratio (width much greater than depth), the EOF can be considered as a two-dimensional gradient shear flow. By using a scanning confocal fluorescence microscope, we demonstrated that negatively charged DNA molecules were focused by the nonuniform EOF and the electric field into a thin layer at the glass surface of about one-fifth of the channel depth. This phenomenon was applied to selectively detect target DNA molecules using evanescent-field excitation. Streptavidin-conjugated quantum dots (QD) were used to selectively bind biotinylated DNA. After applying an electric field, free QD remained randomly distributed within the channel. Nonbiotinylated DNA molecules were focused onto the glass surface, but they were not labeled and thus nonfluorescent. Therefore, the increase in molecule count after application of the electric field can be solely attributed to QD-DNA complexes which were focused onto the glass surface. This method does not require the separation of excessive probes (QD) and can be operated continuously to achieve high throughput.