Live imaging of living cells in micro and nano environment with three-dimensional (3D) architectures is highly demanded for many biological studies. The important issue for distortion-free imaging is refractive index matching between the imaging platform and the culture medium (water). Fluoropolymer CYTOP (AGC Corporation) is a promising material for this application because its refractive index matching (1.34) is almost comparable to water (1.33). However, its excellent properties such as high transparency in an ultraviolet region, high chemical resistance, and water repellence make it very difficult to achieve microfabrication. In this study, we have developed a new 3D fabrication method for CYTOP by using molds fabricated by femtosecond laser two-photon polymerization (2PP). Specifically, a 3D microstructure was first fabricated by two-photon polymerization (2PP) using a femtosecond laser (wavelength: 515 nm, pulse width: 220 fs) with SU8, a photosensitive resin, drop-casted on a cover glass. After the 2PP process, the fabricated 3D structure was used to mold the 3D structure in liquid CYTOP. Finally, the 3D SU8 microstructure was dissolved using piranha solution, leaving the 3D CYTOP microstructure. This technique allowed us to create 3D microfluidic structures inside CYTOP with various widths from sub µm to several tens µm. The fabricated 3D micro/nanofludic structures were applied to live imaging of living cells, showing high-resolution images even near the side walls of micro/nanofluidic channels.