Optically-flat glass channels were fabricated for the non-fluorescent observation of bio-molecules. Two bioassays of motor proteins were successfully performed, in which nanoscale beads and microtubules were clearly observed by a differential interference contrast (DIC) microscope and a dark-field microscope, respectively. The concentration of hydrofluoric acid (HF) was optimized to obtain an optically flat surface which was evaluated by AFM, SEM and dark-field microscopy. A glass channel was etched using a poly(dimethyl siloxane) (PDMS) microfluidic channel as a mask and sealed with a PDMS-coated coverslip. The channel volume of 2–3 µl realized the drastic reduction of proteins required per assay compared with a conventional flow cell method requiring 20 µl.