A microwell arrry device with a hybrid structure of amorphour perfluoroplymer, CYTOP™, and polydimethylsiloxane (PDMS) which is specialized in total internal refllection fluorescence (TIRF) microscopy was developed. Since the CYTOP layer, whose refractive index is as same as that of water, is sandwiched between PDMS and glass substrate, leakage of incident light is avoided. PDMS layer upon the CYTOP enables high aspect ratio structures and easy integration of microfluidic components. Since direct bonding of CYTOP and PDMS is difficult to achieve, a chemical bonding method using aminosilane coupling reagent and 3-Glycoidoxypropyltriethoxysilane was applied. The bond strength was about 0.74 MPa. 24 × 50 microwell array was fabricated and their optical properties was evaluated. Measured TIRF microscopy results shows no interfere with incident light under the conventionally used conditions.