The study presents a direct process for surface patterning of multilayer graphene on the glass substrate as a biosensing device. In contrast to lithography with etching, the proposed process provides simultaneous surface patterning of multilayer graphene through nanosecond laser irradiation. In this study, the multilayer graphene was prepared by a screen printing process. Additionally, the wavelength of the laser beam was 355 nm. To perform the effective laser process with the small heat affected zone, the surface patterns on the sensing devices could be directly fabricated using the laser with optimal control of the pulse overlap at a fluence threshold of 0.63 J/cm(2). The unique patterning of the laser-ablated surface exhibits their electrical and hydrophilic characteristics. The hydrophilic surface of graphene-based sensing devices was achieved in the process with the pulse overlap of 90%. Furthermore, the sensing devices for controlling the electrical response of glucose by using glucose oxidase can be used in sensors in commercial medical applications. (C) 2015 Elsevier B.V. All rights reserved.