In this work a novel gold-based microelectrode was successfully fabricated using photolithographic techniques and electrochemical deposition for a simultaneous determination of nitrate and dissolved oxygen (DO) under neutral conditions. Three-dimensional tree-shaped silver nanorods were formed on the gold surface through electrochemical deposition and they had an electrochemical catalytic reductive activity for both nitrate and oxygen under neutral conditions. Thus, the silver nanorods served as the active center of the microelectrode. The microelectrode could be renewed over five times. Linear sweep voltammetry was employed to quantitatively analyze the nitrate and DO in solution. The microelectrode was used to measure the nitrate and DO microprofiles in a nitrifying aerobic granule from a sequencing batch reactor, which shows that denitrification did not occur in the tested granule. The measurement results demonstrate that the microelectrode was able to simultaneously determine the nitrate and DO levels in the granules under neutral conditions accurately and precisely.