Transdermal biosensors for the real-time and continuous detection and monitoring of target molecules represent an intriguing pathway for enhancing health outcomes in a cost-effective and non-invasive fashion. Many transdermal biosensor devices contain microneedles and other miniaturized components. There remains an unmet clinical need for microneedle transdermal biosensors to obtain a more accurate, rapid, and reliable insight into the real-time monitoring of disease. The ability to monitor biomarkers at an intradermal molecular level in a non-invasive manner remains the next technological gap to solve real-world clinical problems. The emergence of the two-dimensional material graphene with unique material properties and the ability to quantify analytes and physiological status can enable the detection of critical biomarkers indicative of human disease. The development of a user-friendly, affordable, and non-invasive transdermal biosensing device for continuous and personalized monitoring of target molecules could be beneficial for many patients. This focus article considers the use of graphene-based transdermal biosensors for health monitoring, evaluation of these sensors for glucose and hydrogen peroxide detection via in vitro, in vivo, and ex vivo studies, recent technological innovations, and potential challenges. This article is categorized under: Diagnostic Tools > Biosensing.