Abstract Portable biosensors mainly focus on detecting biomarkers in biofluids but neglect the abundant skin biomarkers on the stratum corneum, which are associated with the functionality and integrity of the skin barrier. Here, we propose a sensing patch designed for direct sampling and in situ quantification of epidermal serine, an important biomarker for skin healthcare. The patch consists of a porous hydrogel for serine diffusion and ion conduction, and a molecular imprinted polymer-based electrochemical serine sensor. By integrating with a customized handheld serine tester, the serine sensing system enables in situ measurement of epidermal serine levels. We demonstrate the application of this serine sensing system in assessing the moisturizing effect of a skincare product and tracking the recovery progress of skin barrier function in a patient with atopic dermatitis. Our work opens up a potential application scenario for portable biosensors in personalized skin healthcare.