This paper reports a novel three-dimensional (3D) microelectrode to enhance the sensitivity and current output of the amperometric microsensor. Based on silicon bulk micromachining technology and the introduction of nanomaterials, the 3D microelectrodes (3DME) are fabricated as working electrodes of the amperometric sensor. It comprises both sensitive microstructures and platinum (Pt) nanoparticles. The 3D micro structure can enlarge the effective surface area of working electrode and make more analyte to approach electrode surface more easily. This design provides a better microenvironment for electrodeposition of platinum nanoparticales. It further improves the enhancement effect of catalytic efficiency and electroactivity for the microelectrode. The sensor has been successfully used to detect H2O2, which is a vital target analyte in glucose detection and enzyme-linked immunodetection. Compared with amperometric biosensor based on planar microelectrode (PME), this sensor has advantages of lower detection limit, higher current signal and higher sensitivity.