Chronic diseases (CDs) are noncommunicable illnesses with long-term symptoms accounting for similar to 70% of all deaths worldwide. For the diagnosis and prognosis of CDs, accurate biomarker detection is essential. Currently, the detection of CD-associated biomarkers is employed through complex platforms with certain limitations in their applicability and performance. There is hence unmet need to present innovative strategies that are applicable to the point-of-care (PoC) settings, and also, provide the precise detection of biomarkers. On the other hand, especially at PoC settings, microneedle (MN) technology, which comprises micron-size needles arranged on a miniature patch, has risen as a revolutionary approach in biosensing strategies, opening novel horizons to improve the existing PoC devices. Various MN-based platforms have been manufactured for distinctive purposes employing several techniques and materials. The development of MN-based biosensors for real-time monitoring of CD-associated biomarkers has garnered huge attention in recent years. Herein, we summarize basic concepts of MNs, including microfabrication techniques, design parameters, and their mechanism of action as a biosensing platform for CD diagnosis. Moreover, recent advances in the use of MNs for CD diagnosis are introduced and finally relevant clinical trials carried out using MNs as biosensing devices are highlighted. This review aims to address the potential use of MNs in CD diagnosis.