Low-cost and facile preparation of high-performance nanomaterials has aroused research passion to build nonenzymatic glucose sensor. In this work, uniform and dense Cu nanoparticles were immobilized on ITO electrode by one-step electrochemical deposition, where the Cu nanoparticles/ITO (Cu NPs/ITO) electrode were directly employed for glucose sensing without additional binder. During the deposition, the complexing agent and deposition potential were adjusted to realize the morphology-controlled of Cu NPs and further decrease the aggregation of nanoparticles. Subsequently, the concentration of sodium citrate, deposition potential, NaOH concentration and detection potential on the electrooxidation performances of Cu NPs/ITO were investigated systematically by electrochemical analysis. Under the optimal conditions, the sensor exhibited a high sensitivity of 1005.09 mu A mM(-1) cm(-2) and a wide linear range of 0.0033-3.9019 mM (R-2 = 0.998), benefiting from the high conductivity, large surface area and directly electron transfer of binder-free Cu nanoparticles with uniform and dense morphology. Great stability, reproducibility, selectivity and feasible human serum analysis of Cu NPs/ITO were also demonstrated in glucose sensing. Owing to its low-cost, one-step preparation and great performance, this Cu NPs/ITO electrode can be a promising candidate for the construction of non-enzymatic glucose sensor.