An integrated system was fabricated from a copper(II) metal–organic framework (Cu-MOF) and glucose oxidase (GOx) for one-step cascade determination of glucose at neutral pH (pH = 7.0). Herein, the Cu-MOF performs dual functions of loading GOx and mimicking peroxidase. The GOx@MOF integrated composites were synthesized under mild conditions. In the integrated system, glucose was oxidized to gluconic acid and hydrogen peroxide by GOx in the presence of oxygen. In the presence of the MOF catalyst, hydrogen peroxide, acting as an intermediate product, was immediately converted to ˙OH, which oxidized 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and UV-vis absorbance at 420 nm was observed. The glucose concentration is positively and linearly correlated with absorbance in the range 8–140 μM with a detection limit of 2.67 μM. Our strategy possesses the characteristics of excellent accuracy, easy operation and biological application. It has been successfully applied to the detection of glucose in human serum, and provides a more convenient and sensitive operation platform for biological analysis.