In this work, a new micro-needle sensor based on copper microspheres (CuMSs) and polyaniline (PANI) film was fabricated through electrodeposition method and used for the voltammetric determination of nitrate (NO −3 ) in coastal river samples. The PANI film functions as the conductive polymer and adhesion agent to immobilize the CuMSs which has good electrocatalytic property for the reduction of NO − 3 . The CuMSs could be more easily deposited on the micro-needle electrode (MNE) surface with PANI film as adhesion agent. The physical and electrochemical properties of the as-prepared micro-needle electrode were characterized by different techniques. CuMSs with diameter of about 0.5 to 1 μm were decorated on the PANI film modified micro-needle electrode. The unique structure of micro-needle electrode and excellent properties of CuMSs and PANI film make the micro-needle sensor possesses the advantages of larger specific surface and high electrocatalytic property towards the reduction of NO − 3 . The micro-needle electrode based on CuMSs and PANI film shows a linear response to NO − 3 in the concentrations ranging from 0.02 to 6 mM (R 2 = 0.995) with the detection limit of 8 μM. More importantly, the determination of NO − 3 in coastal river water samples was achieved with the micro-needle electrode with satisfactory results.