This study describes a type of novel nickel nanoparticles (NiNPs) decorated on Nafiongraphene composite film by using the electrochemical deposition method. It was used to fabricate electrochemical biosensors for sensitive nonenzymatic glucose detection. Compared with the Nafion-graphene film and NiNPs-modified glassy carbon electrode (NiNPs-GCE), the NiNPs/Nafion/graphene/GCE showed the best electrocatalytic activity towards glucose oxidation in alkaline medium. The NiNPs/Nafion/graphene/GCE at an applied potential of +0.55 V in a linear range of 1-200 mu M presented a high sensitivity of 3437.25 mu A center dot mM(-1) cm(-2) with coefficient of correlation R-2 = 0.999; and in a linear range of 200-10800 mu M it performed the best sensitivity of 2848.6 mu A center dot mM(-1) cm(-2) with coefficient of correlation R-2 = 0.995 towards glucose oxidation. For a concentration up to 200 mu M, a linear range was obtained with a limit of detection of 0.6 mu M (signal to noise = 3) and as much as 10 800 mu M with a limit of detection of 0.82 mu M (signal to noise = 3). The time of responses was about 1-1.5 s with the addition of 0.1-1 mM glucose. In addition, NiNPs/Nafion/graphene/GCE also has a high anti-interference ability toward common oxidative interfering species, such as uric acid, ascorbic acid and dopamine. More importantly, NiNPs/Nafion/graphene/GCE was successfully used for the determination of glucose concentration in human serum samples in comparison with a local hospital. The NiNPs/Nafion/graphene/GCE exhibited high sensitivity, low working potential, good stability, excellent electrical properties, enhanced selectivity and fast amperometric responses to glucose oxidation. Thus, as a nonenzymatic sensor, it is promising for future glucose determination development.