This study describes a type of novel nickel nanoparticles (NiNPs) decorated on Nafion-graphene 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 [Formula: see text]0.55[Formula: see text]V in a linear range of 1–200[Formula: see text][Formula: see text]M presented a high sensitivity of 3437.25[Formula: see text][Formula: see text]A[Formula: see text]mM[Formula: see text][Formula: see text]cm[Formula: see text] with coefficient of correlation [Formula: see text]; and in a linear range of 200–10[Formula: see text]800[Formula: see text][Formula: see text]M it performed the best sensitivity of 2848.6[Formula: see text][Formula: see text]A[Formula: see text]mM[Formula: see text][Formula: see text]cm[Formula: see text] with coefficient of correlation [Formula: see text] towards glucose oxidation. For a concentration up to 200[Formula: see text][Formula: see text]M, a linear range was obtained with a limit of detection of 0.6[Formula: see text][Formula: see text]M (signal to noise [Formula: see text] 3) and as much as 10[Formula: see text]800[Formula: see text][Formula: see text]M with a limit of detection of 0.82[Formula: see text][Formula: see text]M (signal to noise [Formula: see text] 3). The time of responses was about 1–1.5[Formula: see text]s with the addition of 0.1–1[Formula: see text]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.