The field of portable healthcare monitoring devices has an urgent need for the development of real‐time, noninvasive sensing and detection methods for various physiological analytes. Currently, transdermal sensing techniques are severely limited in scope (i.e., measurement of heart rate or sweat composition), or else tend to be invasive, often needing to be performed in a clinical setting. This study proposes a minimally invasive alternative strategy, consisting of using dissolving polymeric microneedles to deliver naked eye‐invisible functional fluorescent ratiometric microneedle tattoos directly to the skin for real‐time monitoring and quantification of physiological and pathological parameters. Reactive oxygen species are overexpressed in the skin in association with various pathological conditions. Here, one demonstrates for the first time the microneedle‐based delivery to the skin of active fluorescent sensors in the form of an invisible, ratiometric microneedle tattoo capable of sensing reactive oxygen species in a reconstructed human‐based skin disease model, as well as an in vivo model of UV‐induced dermal inflammation. One also elaborates a universal ratiometric quantification concept coupled with a custom‐built, multiwavelength portable fluorescence detection system. Fully realized, this approach presents an opportunity for the minimally invasive monitoring of a broad range of physiological parameters through the skin.