The organ-on-a-chip (OOAC) is in the list of top 10 emerging technologies and refers to a physiological organ biomimetic system built on a microfluidic chip. Through a combination of cell biology, engineering, and biomaterial technology, the microenvironment of the chip simulates that of the organ in terms of tissue interfaces and mechanical stimulation. This reflects the structural and functional characteristics of human tissue and can predict response to an array of stimuli including drug responses and environmental effects. OOAC has broad applications in precision medicine and biological defense strategies. Here, we introduce the concepts of OOAC and review its application to the construction of physiological models, drug development, and toxicology from the perspective of different organs. We further discuss existing challenges and provide future perspectives for its application.