Hollow spheres have shown fascinating application in bionanotechnology recently, including bioanalysis, diagnostics, drug delivery and therapeutics. However the exploration of hollow spheres via template- and surfactant-free synthesis and using them as sensing material is still at an early stage. In this regard, we described a novel solution-based chemical process to fabricate Ni(OH)2 hollow spheres (Ni(OH)2-HS) assembled from nanosheet building blocks. Ni(OH)2-HS can be obtained with this template-free approach under one-step hydrothermal method. Various techniques, including X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), transmission electron microscopy (TEM), field emission scanning electron microscopy (FE-SEM), were used to characterize the morphology and the structure of the as-prepared samples. It was confirmed that the products possess a hollow microsphere structure constructed by interconnecting nanosheet framework. The as-obtained hierarchical structured Ni(OH)2-HS showed excellent catalytic activity toward the oxidation of glucose in alkaline solutions, which enables the Ni(OH)2-HS to be used in enzyme-free amperometric sensors for glucose determination. Furthermore, Ni(OH)2-HS modified glassy carbon electrode (Ni(OH)2-HS/GCE) exhibited a good ability to suppress the background current from large excess ascorbic acid (AA), uric acid (UA) and L-cysteine. Under the optimal conditions, selective detection of glucose in a linear concentration range of 0.8749μM-7.781mM was obtained with the limit of 0.1μM (S/N=3). Meanwhile, the sensors were also applied to the detection of glucose content in real serum sample with satisfactory results.