The rapid growth of flexible electronics has led to various wearable sensors, circuit, and storage devices. The glucose biofuel cell (GFC) is a device capable of converting the chemical energy from glucose fuel into electrical energy, with a wide range of applications in self-powered wearable medical devices. In this study, a PtAu nanoparticle catalyst was synthesized for the catalysis of glucose in a neutral environment through an ultrasound-assisted method. This method offers the advantages of simple operation, short reaction time, and environmental friendliness. In the synthesis of PtAu catalyst, drying is unnecessary, but the catalyst concentration must be carefully controlled to maintain its catalytic effectiveness. The morphology and structure of the PtAu catalysts were analyzed using transmission electron microscopy (TEM), X-ray diffraction (XRD), and X-ray photoelectron spectrum (XPS). The PtAu catalysts were immobilized on a carbon cloth (CC) to construct the anode of flexible GFC. The study investigated the impact of electrochemically active area, varying sweep rates, and pH values on the catalytic performance, demonstrating the PtAu catalyst’s commendable activity and durability for glucose catalysis. Furthermore, a solid manganese dioxide (MnO₂) material was synthesized through a simple hydrothermal method. The morphology and structure MnO₂ were characterized using scanning electron microscopy (SEM) and XRD. The prepared solid MnO₂ material was applied to the CC as the cathode of flexible GFC to address issues such as low oxygen solubility and slow O₂ reduction kinetics in solution. Linear scanning voltammetry (LSV) was employed to examine the influence of glucose on MnO₂. In addition, the effects of various mechanical deformations on the anode and cathode were studied, demonstrating their strong mechanical strain capacity. A membrane-less flexible GFC prepared using a PtAu/CC anode and a MnO₂/CC cathode achieved a maximum power density (P_max) of 22.61 μW•cm⁻² and an open circuit voltage (E^OCV) of 0.439 V, surpassing previous reports in the literature, demonstrating its potential for wearable applications. Notably, the flexible GFC maintains consistent power density and E^OCV even after repeated bending, highlighting its stability.