Currently, most metal oxides- or hydroxides-based and non-enzymatic sensors require a high alkali concentration in the working environment. Here, we designed a novel non-enzymatic sensor based on a 3D nanostructured Co(OH)₂/rGO film to address this issue. The architecture of the hybrid film was in-situ constructed by the dense assembly of 200 nm flower-like Co(OH)₂ crystals bridged by rGO nanosheets. This structure possessed an extremely large specific surface area and an excellent conductivity which were beneficial for adsorbing OHsingle bond to produce oxidative CoOOH or CoO₂, even in a low alkaline condition. This material was able to mimic the functionality of glucose oxidase to oxidize glucose to gluconolactone and exhibited a good sensitivity at a wide alkaline range from 5 × 10⁻³ to 1 mol L⁻¹ NaOH solution. Moreover, this sensor showed a wide linear range, excellent selectivity, reproducibility and stability for real blood analysis.