This paper describes the fabrication and characterization of copper nano-clusters prepared by a simple one-step electrodeposition process on platinum microelectrode, and the application for nitrate determination. The one-step electrodepostion process was performed by chronoamperometry scan in acidic copper sulphate electrolyte directly. The SEM and electrochemical examination showed that the morphologies and microstructures of deposited copper layers can be precisely controlled by using different deposition voltages. It was found that the copper layer is porous when the deposition voltage is higher than -500 mV, and this porous layer has a larger effective surface area compared with the corresponding smooth flat copper layer deposited under voltage less than -300 mV. Under the optimized deposition voltage, copper clusters constructed by uniform nanoparticles with an average diameter of about 100 nm can be obtained. The mechanism of electrodeposition process for this method was also speculated. The copper layers deposited under different voltages are used in a series of tests in order to evaluate their performance for nitrate sensing. The experimental results reveal that the microelectrode modified by fixed potential deposition under -700 mV had a higher sensitivity of 39.31 μA/mmolL−1 for nitrate detection within the concentration ranging from 0.1 mmolL−1 to 4.0 mmolL−1.