In this study, we develop a new technique to fabricate a reduced graphene oxide (rGO)-based microelectrode array (MEA) with low-cost soft lithography. To prepare patterned rGO, a polydimethylsiloxane (PDMS) mold with an array of microwells on its surface is fabricated using soft lithography, and GO is assembled on an indium tin oxide (ITO) electrode with a layer-by-layer method. The rGO pattern is formed by closely contacting the assembled GO film onto the ITO electrode with the PDMS mold filled with hydrazine solution in the microwells to selectively reduce the localized GO into the rGO. The MEA with patterned rGO as the microelectrode is characterized with Kelvin probe force microscopy (KFM), atomic force microscopy (AFM), and cyclic voltammetry (CV) with ferricyanide in aqueous solution as the redox probe. The KFM and AFM results demonstrate that each rGO pattern prepared under the present conditions is 3 μm in diameter, which is close to that of the PDMS mold we use. The CV results show that the rGO patterned onto the ITO exhibits a sigmoid-shaped voltammogram up to 200 mVs(-1) with a microampere level current response, suggesting that the rGO-based electrode fabricated with soft lithography behalves like a MEA. To demonstrate the potential electroanalytical application of the rGO-based MEA, prussian blue (PB) is electrodeposited onto the rGO-based MEA to form the PB/rGO-based MEA. Electrochemical studies on the formed PB/rGO-based MEA reveal that MEA shows a lower detection limit and a larger current density for the detection of H(2)O(2), as compared with the macroscopic rGO electrode. The method demonstrated here provides a simple and low-cost strategy for the fabrication of graphene-based MEA that are useful for electroanalytical applications.