The development of non-invasive biosensor for monitoring glucose in interstitial fluid (ISF) is still challenging, because ISF extraction through classical reverse iontophoresis (RI) is limited by low extraction flux and consistency. Here, we developed a touch-actuated biosensor for monitoring glucose in ISF. The biosensor is composed of three main components: 1) the solid microneedle array (MA) for painless skin penetration; 2) the RI unit for ISF extraction through the MA-created microchannels; and 3) the sensing unit for glucose monitoring. The sensing strategy of this biosensor is “skin penetration-RI extraction-electrochemical detection”. Compared with RI extraction only, the reported skin penetration-RI extraction sampling strategy obviously increased the glucose extraction flux by ∼1.6 times not only in vitro but also in vivo. Moreover, we developed a wearable glucose monitoring system by incorporating this touch-actuated biosensor, a wireless electrochemical detector, and a smartphone application. In vivo experiments using healthy and diabetic rats revealed a high correlation between the results measured by the reported wearable system and commercially blood glucometer. This sampling strategy which combined skin penetration and RI extraction paves the way to develop wearable platforms for not only glucose monitoring but also various ISF biomarkers without the need of painful finger-stick blood sampling.