Diabetes remains a great threat to human beings’ health and its world prevalence is projected to reach 9.9% by 2045. At present, the detection methods used are often invasive, cumbersome and time-consuming, thus increasing the burden on patients. In this paper, we propose a novel noninvasive and low-cost biosensor capable of detecting glucose in human sweat using enzyme-based electrodes for point-of-care uses. Specifically, an electrochemical method is applied for detection and the electrodes are covered with multilayered films including ferrocene-polyaniline (F-P), multi-walled carbon nanotubes (MWCNTs) and glucose oxidase (GOx) on Cu substrates (GOx/MWCNTs/F-P/Cu). The coated layers enhance the immobilization of GOx, increase the conductivity of the anode and improve the electrochemical properties of the electrode. Compared with the Cu electrode and the F-P/Cu electrode, a maximum peak current is obtained when the MWCNTs/F-P/Cu electrode is applied. We also study its current response by cyclic voltammetry (CV) at different concentrations (0–2.0 mM) of glucose solution. The best current response is obtained at 0.25 V using chronoamperometry. The effective working lifetime of an electrode is up to 8 days. Finally, to demonstrate the capability of the electrode, a portable, miniaturized and integrated detection device based on the GOx/MWCNTs/F-P/Cu electrode is developed. The results exhibit a short response time of 5 s and a correlation coefficient R2 of 0.9847 between the response current of sweat with blood glucose concentration. The LOD is of 0.081 mM and the reproducibility achieved in terms of RSD is 3.55%. The sweat glucose sensor is noninvasive and point-of-care, which shows great development potential in the health examination and monitoring field.