Biosensors have attracted a considerable attention in recent years due to their enormous potential to provide insights into the physical condition of individuals. However, the widespread use of biosensors has experienced difficulties regarding the stability of the biological response and the poor miniaturization and portability of biosensors. Hence, there is an urgent need for more reliable biosensor devices. Microneedle (MN) technology has become a revolutionary approach to biosensing strategies, setting new horizons for improving existing biosensors. MN-based biosensors allow for painless injection, and in situ extraction or monitoring. However, the accuracy and practicality of detection need to be improved. This review begins by discussing the classification of MNs, manufacturing methods and other design parameters to develop a more accurate MN-based detection sensing system. Herein, we categorize and analyze the energy supply of wearable biosensors. Specifically, we describe the detection methods of MN biosensors, such as electrochemical, optical, nucleic acid recognition and immunoassays, and how MNs can be combined with these methods to detect biomarkers. Furthermore, we provide a detailed overview of the latest applications (drug release, drug detection, etc.). The MN-based biosensors are followed by a summary of key challenges and opportunities in the field.