We show that fabrication of three-dimensional microfluidic channels embedded in glass can be achieved by using a Q-switched, frequency-doubled Nd:YAG laser. The processing mainly consists of two steps: (1) formation of hollow microfluidic channels in porous glass immersed in Rhodamine 6G dissolved in water by nanosecond laser ablation; and (2) postannealing of the fabricated porous glass sample at 1120 °C for consolidation of the sample. In particular, a bilayer microfluidic structure is created in glass substrate using this technique for showcasing its capability of three-dimensional structuring.