We fabricated a glucose biosensor based on glucose oxidase (GOx) immobilized in a poly(L-lysine) (PLL) and reduced graphene oxide–zirconium oxide composite (RGO–ZrO₂). First, a simple single step electrochemical approach was used to prepare the RGO–ZrO₂ composite and its successful formation was confirmed by various techniques. Then, a mixture of GOx and PLL was immobilized to form the RGO–ZrO₂ composite. Direct electrochemistry of the GOx was attained for the composite film showing well-defined redox peaks centered at the formal potential −0.403 V. Notably, the peak to peak separation (ΔE_p) was very low (27 mV), while the heterogeneous rate constant for the fast electron transfer was calculated to be 5.03 (±0.14 s⁻¹) revealed. The composite film modified electrode exhibited excellent electrocatalytic ability through the reductive detection of oxygen. A glucose biosensor was developed using the GOx–PLL/RGO–ZrO₂ composite film modified electrode which functioned using differential pulse voltammetry (DPV). The fabricated biosensor exhibited good electrocatalytic ability for the determination of glucose with excellent analytical parameters such as wide linear range of 0.29 mM to 14 mM and high sensitivity of 11.65 (±0.17) μA mM⁻¹ cm⁻². The sensor results also exhibited appreciable repeatability, reproducibility and stability. Good recovery rates were achieved for the real sample studies proving the promise and practicality of the proposed sensor.