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Over the past decade, the development of non-enzymatic electrochemical biosensors had thriven at a considerable rate. Compared with the traditional enzymatic electrochemical biosensors, the non-enzymatic electrochemical biosensors have the advantages of higher sensitivity and stability. Recently, plenty of researches have devoted to synthesizing new materials, such as bimetallic nanoparticles, and also develop specific nanostructures on the sensor surface to solve the problem of poisoning and increase the selectivity. This work develops two non-enzymatic glucose sensors that are based on nanostructured Pt–Ir films which were deposited by electrodeposition. Because of the relatively high deposition current density, bubbles produced vigorously on the working electrode surface. This phenomenon results in leaf-like nanostructure formed naturally on the surface of the working electrode and further increased the catalytic reaction area. Besides, as determined by the sampling analysis method that is developed herein, the presented Pt–Ir sensors mitigate the current drifting problem which is easily observed when a constant potential is applied in an amperometric glucose detection. Furthermore, the presented Pt–Ir sensors show high sensitivity and stability in 1X PBS (0.15 M NaCl) at 37°C in the glucose concentration range of 1–12 mM. Therefore, the presented non-enzymatic glucose sensors not only provide great potential in biomedical applications, such as homecare products, but can also be adapted for the biological application, such as continuous cell culture monitoring.