We present surface microfabrication of polyacrylamide (PAA) hydrogel using a femtosecond laser. Hydrogels have been widely used in the fields of tissue engineering and organ-on-a-chip due to their high flexibility, biocompatibility and so on. Among many gels, PAA has numerous useful characteristics, including high optical transmittance, low refractive index similar to that of water, and low cost. Therefore, it can be one of candidates for biochip materials. In this study, we investigated on the microfabrication of low refractive index PAA using fs laser direct writing for biochip applications. As an experiment to find out, the laser irradiation in the pure water and in dried samples were used to produce the microgroove. These methods were taken to prevent shape changes due to progressive drying. The laser irradiation was utilized from a Yb:KGW laser (wavelength 1035 nm, pulse width 100 fs, repetition rate 75.6 MHz). The laser pulse was focused on the PAA surface through an objective lens (×20, NA:0.75). The microgroove fabricated by the fs laser ablation in water has sharp edge with low ablation depth compare to the microgroove fabricated on the dried PAA with successive swelling in water. In this presentation, we investigate the fundamental characteristics of the fs laser microfabrication of PAA in detail.