Thermophoresis refers to the directional movement of particles driven by temperature gradients, which is ubiquitous in nature. This phenomenon can be conveniently used to manipulate molecules and objects such as biomolecules, colloids, metal nanoparticles, and cells. Because of these advantages, thermophoresis is widely used in biosensing. In vitro diagnosis, as an important field of biosensing, is highly compatible with thermophoresis. Compared with traditional in vitro diagnostic techniques, thermophoretic diagnosis is a homogeneous system that boasts high efficiency and rapid detection as well as a wide detection range; thus, thermophoresis has a myriad of applications. The emergence of microscale and multifield coupling thermophoresis technologies yield widespread applications of thermophoresis in the field of in vitro diagnostics. This study presents the history of thermophoresis and reviews various cases of biosensors utilizing thermophoresis. Those biosensors are widely used in in vitro diagnostics, biomarker discovery, and other fields. Furthermore, it assesses the advantages of thermophoresis, and finally discusses its current challenges and future development.