Abstract The direct electron transfer (DET) between glucose oxidase (GOD) and the underlying glassy carbon electrode (GCE) can be readily achieved via colloidal laponite nanoparticles as immobilization matrix. Cyclic voltammetry of laponite/GOD/GCE, in anaerobic phosphate buffer solution (PBS, 0.1 M, pH 5.0), showed a pair of stable and quasi-reversible peaks at potentials E pa = −0.372 V and E pc = −0.391 V vs. SCE, provoked by the prosthetic FAD group linked to the protein. The electrochemical reaction of laponite/GOD/GCE exhibited a surface-controlled process with the apparent heterogeneous electron transfer rate constant ( k s) of 6.52 s −1 and charge-transfer coefficient ( α) of 0.5. The experiments of FTIR and UV–vis spectroscopy demonstrate that the immobilized GOD on colloidal laponite nanoparticles retained its native structure and its biocatalytic ability to its substrates. Based on the decrease of oxygen electrocatalytic signal, the proposed laponite/GOD/GCE was successfully applied in the reagentless glucose sensing at −0.45 V. The proposed electrode exhibited fast amperometric response (8 s), broad linear range (2.0 × 10 −5–1.9 × 10 −3 M), good sensitivity (4.8 ± 0.5 mA M −1 cm −2), low detection limit (1.0 × 10 −5 M) at a signal-to-noise ratio of 3, and excellent selectivity.