Herein, we report a non-enzymatic electrochemical glucose sensing platform based on NiS nanoclusters dispersed on NiS nanosphere (NC-NiS@NS-NiS) in human serum and urine samples. The NC-NiS@NS-NiS are directly grown on nickel foam (NF) (NC-NiS@NS-NiS|NF) substrate by a facile, and one-step electrodeposition strategy under acidic solution. The as-developed nanostructured NC-NiS@NS-NiS|NF electrode materials successfully employ as the enzyme-mimic electrocatalysts toward the improved electrocatalytic glucose oxidation and sensitive glucose sensing. The NC-NiS@NS-NiS|NF electrode presents an outstanding electrocatalytic activity and sensing capability towards the glucose owing to the attribution of great double layer capacitance, excessive electrochemical active surface area (ECASA), and high electrochemical active sites. The present sensor delivers a limit of detection (LOD) of ~0.0083 µM with a high sensitivity of 54.6 µA mM−1 cm−2 and a wide linear concentration range (20.0 µM–5.0 mM). The NC-NiS@NS-NiS|NF-based sensor demonstrates the good selectivity against the potential interferences and shows high practicability by glucose sensing in human urine and serum samples.