The development of nanomaterials with notable enzyme-like activity is of great significance to the field of bioanalysis. Herein, a nanomaterial composed of copper ferrite nanoparticles (CuFe2O4 NPs) and graphene oxide (GO) was successfully prepared (CuFe2O4@GO). The obtained CuFe2O4@GO was proved to have peroxidase-like (POD-like) activity, which could catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to generate oxidized TMB (ox-TMB) in the presence of H2O2 with an obvious color change. On this basis, a colorimetric biosensor using a UV–vis spectrometer as readout and a paper chip-based colorimetric biosensor using a smartphone as readout was constructed for glucose detection, respectively, as glucose oxidase (GOD) could oxidize glucose to form H2O2. The UV–vis spectrometer-based approach exhibits a linear range of 0 to 1000 μM and a limit of detection (LOD) of 0.79 μM for glucose. The smartphone-based method shows a linear range of 0.1 to 50 mM and a LOD of 0.04 mM for glucose. Moreover, the fabricated colorimetric biosensors have good selectivity towards glucose and the sample matrixes cannot affect the detection of glucose. Finally, the concentration of glucose in human serum samples was successfully detected using two different ways, and the results are close to the clinical values. The proposed method embraces several good merits. Firstly, the colorimetric biosensors combining UV–vis spectrometer and smartphone offer precise results, which could effectively prevent the occurrence of false events. Secondly, paper-based microfluidic analytical device (μPAD) as analytical platform and smartphone as readout provides a versatile approach for constructing sensitive, cost-effective, and simple colorimetric biosensor for the visual monitoring of glucose. Lastly, except for glucose detection, this work also shows great application prospects for other small molecules detection that could generate H2O2.