Janus particles (JPs), named after the two-faced Roman god, are asymmetric particles with different chemical properties or polarities. JPs have been widely used in the biomedical field in recent years, including as drug carriers for targeted controlled drug release and as biosensors for biological imaging and biomarker detection, which is crucial in the early detection and treatment of diseases. In this review, we highlight the most recent advancements made with regard to Janus particles in point-of-care testing (POCT). Firstly, we introduce several commonly used methods for preparing Janus particles. Secondly, we present biomarker detection using JPs based on various detection methods to achieve the goal of POCT. Finally, we discuss the challenges and opportunities for developing Janus particles in POCT. This review will facilitate the development of POCT biosensing devices based on the unique properties of Janus particles.