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The synthesized NiCo 2 O 4 mesoporous spheres (MS) displayed intrinsic peroxidase and oxidase-like activity were firstly reported. The catalytic mechanism of the oxidase-like activity of NiCo 2 O 4 MS was analyzed in detail using the electron spin resonance (ESR) method. It is found that NiCo 2 O 4 MS could directly oxidize 3,3′,5,5′-tetramethylbenzidine (TMB) but did not produce 1 O 2 and ·OH. And the mechanism of the peroxidase-like activity of NiCo 2 O 4 MS was also verified that the oxidation of TMB stemmed from not only ·OH but also 1 O 2 . Based on the NiCo 2 O 4 MS showed excellent peroxidase-like activity over a broad temperature range, especially at normal body temperature, a detection tool was designed for glucose determination in diabetics' serum samples. And this detection method based on NiCo 2 O 4 MS gave a lower limit of detection than the method using Co 3 O 4 NPs and NiO NPs, as the single-component oxides of NiCo 2 O 4 . Our study may open up the possibility to make a great influence on the next generation of enzyme mimetics system. [ABSTRACT FROM AUTHOR]