Fabrication of three-dimensional microfluidic channels in glasses by water-assisted ablation with femtosecond laser pulses was investigated. The experimental results showed that formation of the photoinduced microchannels by femtosecond pulses depended on the incident laser power and the scanning speed. For the same scanning speed, the shape of cross-section of channels changed from ellipse to circle with increasing the laser power. Under the optimum condition of laser processing, we fabricated two layers of microfluidic channels with diameter of about 8μm inside glass. The distance between two layers of microchannels was about 20μm.