A PEDOT:PSS conductive hydrogel is incorporated with Prussian blue nanoparticles for the highly sensitive in vitro glucose detection and is applied as a skin patch on human subjects for the in vivo noninvasive monitoring of interstitial fluid (ISF) glucose via reverse iontophoresis technology. The biosensor achieves good correlation with the finger-stick blood glucose measurement. Display omitted •The PEDOT:PSS/DF/PB hydrogel possessed an excellent operating stability.•The hydrogel-based sensor showed a competitive sensitivity of 340.1 μA·mM−1·cm−2.•The wearable sensor noninvasively monitored the ISF glucose through RI-extraction.•The sensor showed good correlation with the results from commerical glucometer. Currently, there is no commercially-available noninvasive method for glucose monitoring in the interstitial fluid (ISF) mainly due to the measurement accuracy, sensitivity and/or the weak blood correlation. Here, we present an electrochemical biosensor based on PEDOT:PSS conductive hydrogel incorporated with Prussian blue nanoparticles (PBNPs) for the noninvasive and continuous monitoring of glucose on body. The hydrogel was prepared with the addition of dimethyl sulfoxide (DMSO) and Zonyl FS-300, which helped to improve the expanding of the polymer chains for the formation of better conductive and nanoporous networks. The hydrogel-based biosensor showed a low detection limit of 0.85 μM for glucose detection and high sensitivity up to 340.1 μA·mM−1·cm−2, which was about 10-fold higher than the counterparts based on agarose and hydroxyethyl methacrylate (HEMA) hydrogels. Furthermore, it provided good accuracy with commercial glucometer for the glucose detection in serum. More importantly, incorporated with the reverse iontophoresis (RI), the hydrogel was applied as a skin patch on human subject for the in vivo noninvasive and continuous monitoring of ISF-borne glucose. The results showed good correlation with that measured by blood glucometer. We believe such glucose biosensor paves the way to clinically wearable noninvasive glucose monitoring in diabetics.