Diabetes and impaired glucose regulation (IGR) threaten the lives and health of numerous patients. Interstitial fluid (ISF) glucose, displaying an excellent correlation with blood glucose, is highly desired to address the limitations of invasive and minimally invasive glucose detection. Herein, we present a screen-printed iontophoretic biosensing system to extract ISF noninvasively and perform in situ instant glucose detection. A three-dimensional graphene aerogel combined with Prussian blue (GA@PB) was introduced as an electron mediator, providing suitable support for glucose oxidase (GO_x) immobilization, highly boosting the detection sensitivity. Additionally, a self-made diffuse cell and an ex vivo model were developed to demonstrate the efficacy of ISF extraction based on reverse iontophoresis technology. Highly sensitive and accurate detection of ISF glucose could be achieved with an LOD of 0.26 mM over a 0–15 mM range. Finally, tests on healthy volunteers were conducted to further validate the feasibility of this as-proposed system. Combined with its well flexible and biocompatible features, it holds considerable prospects in the development of wireless wearable biosensors for continuous blood glucose monitoring.