A low-cost, flexible, and sensitive amperometrical biosensor for the determination of glucose was developed using reduced graphene oxide (rGO) patterns. rGO patterns were created on polyethylene terephthalate (PET) substrates by stamping patterned graphene oxide (GO) using filtration/wax-printing methods and then reducing to rGO to provide conductivity. The resulting biosensor exhibited a fast response time of 0.37 s, a sensitivity of 11.2 μA mM -1 cm -2 , a linear range of 1.0-25.0 mM, and a low detection limit of 0.32 mM. The platform was also found to be reproducible, stable, selective, and resistant to interference. The proposed biosensor can be used for the determination of glucose in human serum, indicating its promising applicability in real sample analysis. Although the biosensor demonstrated high performance, the repeatability and reproducibility showed RSD values of 4.8% and 6.1%, respectively. Overall, the rGO pattern-based amperometrical biosensor provides an effective proof-of-concept for glucose determination and can be applied to different biosensing systems to miniaturize detection instruments.