The forming process of U-form glass micro-nanofluidic chip with long nanochannels is presented in this paper, in which the fabrication of channels and the assembly of plates are included. The micro-nanofluidic chip is composed of two glass plates in which there are microchannels and nanochannels, respectively. This chip can be used for trace sample enrichment, molecule filtration, and sample separation, etc. In fabrication process, the two-step photolithograph on one wafer is often required in early papers, as nano and micro structure designed in one plate have different depths. In this paper, the channels in micro-nanofluidic chip are designed in two glass plates instead of in one wafer. The nanochannels and microchannels are, respectively, formed on plates using wet etching and two-step photolithograph on one wafer is not required. Since the channels are formed, the upper plate and the bottom plate are assembled together by alignment, preconnection and thermal bonding orderly. Firstly these plates are aligned with the cross-marks on an inverted microscope. The aqueous film between plates is controlled to decrease the static friction force for accurate adjustment. Then the adhesion strength of connection is enhanced with semi-dry status for limiting movement from slight inclining and shaking. At last, the bottom plate and the upper one are irreversibly linked together with thermal bonding. The heating period and max temperature of thermal bonding are optimized to eliminate thermal stress gradient and the size shrinking. With the micro-nanofluidic chip, the 1 μM fluorescein isothiocyanate in 10 mM PBS buffer is concentrated successfully. The sample concentrating factor of light intensity varies from 2.2 to 8.4 with applied voltages between 300 and 2,000 V. The switch effect and the instability effect in concentrating process are described and analyzed too.