Internal modification of transparent materials such as glass can be carried out using multiphoton absorption induced by a femtosecond (fs) laser. The fs-laser modification followed by thermal treatment and successive chemical wet etching in a hydrofluoric (HF) acid solution forms three-dimensional (3D) hollow microstructures embedded in photosensitive glass. This technique is a powerful method for directly fabricating 3D microfluidic structures inside a photosensitive glass microchip. We used fabricated microchips, referred to as a nanoaquarium, for dynamic observations of living microorganisms. In addition, the present technique can also be used to form microoptical components such as micromirrors and microlenses inside the photosensitive glass, since the fabricated structures have optically flat surfaces. The integration of microfluidics and microoptical components in a single glass chip yields biophotonic microchips, in other words, optofluidics, which provide high sensitivity in absorption and fluorescence measurements of small volumes of liquid samples.Dynamic observation of microorganisms (Euglena gracilis) using nanoaquarium, which has 3D microfluidic structure, fabricated in a photosensitive glass microchip by femtosecond laser direct writing followed by thermal treatment and successive wet chemical etching.