This study aims to develop graphene-based impedance chips for an electrochemical impedance instrument fabricated by ultraviolet (UV) laser patterning. The finger-like electrode structure on multilayer graphene films coated on glass substrates was performed in a one-cycle laser patterning process. After a series of laser ablated line tests, the ablated line widths increased with increasing laser fluences. The optimal laser patterning parameters involved the laser fluence of 3.4 J/cm2, scanning speed of 600 mm/s, and pulse repetition frequency of 100 kHz to remove completely a graphene electrode layer without damaging glass substrate. The patterned regions had no significant heat-affacted zones (HAZs) due to graphene materials with high thermal diffusivity. To clarify graphene removal completely, the patterned regions on graphene/glass substrates were detected by a Raman microscope. The developed impedance-sensing chips had been successfully applied to detect the phase separation for emulsions containing emulsifiers with 15%, 17.5%, and 20% Polysorbate 80 (Tween 80) concentrations. We found that the separation time increased with increasing the emulsifier concentration.