Abstract: The present invention belongs to the technical field of material fabrication, and particularly relates to an ultra-sensitive glucose sensor based on a graphene and carbon fiber substrate and a fabrication method thereof. The method includes fabricating a carbon fiber cloth with vertical graphene growth on a surface thereof, performing pretreatment to make the carbon fiber cloth hydrophilic, directly soaking the carbon fiber cloth in a PBS solution of glucose oxidase with the pH of 7.4, and then taking out and drying the carbon fiber cloth at room temperature to obtain a glucose sensor. According to the present invention, the lower limit of glucose detection reaches about 0.1 mM, and the glucose sensor also has multistage corresponding characteristics, so that different detection coefficients and capabilities can be achieved in different glucose concentration ranges. The application range and precision of the glucose sensor are greatly improved.