Separation and detection are ubiquitous in our daily life and they are two of the most important steps toward practical biomedical diagnostics and industrial applications. A deep understanding of working principles and examples of separation and detection enables a plethora of applications from blood test and air/water quality monitoring to food safety and biosecurity; none of which are irrelevant to public health. Microfluidics can separate and detect various particles/aerosols as well as cells/viruses in a cost-effective and easy-to-operate manner. There are a number of papers reviewing microfluidic separation and detection, but to the best of our knowledge, the two topics are normally reviewed separately. In fact, these two themes are closely related with each other from the perspectives of public health: understanding separation or sorting technique will lead to the development of new detection methods, thereby providing new paths to guide the separation routes. Therefore, the purpose of this review paper is two-fold: reporting the latest developments in the application of microfluidics for separation and outlining the emerging research in microfluidic detection. The dominating microfluidics-based passive separation methods and detection methods are discussed, along with the future perspectives and challenges being discussed. Our work inspires novel development of separation and detection methods for the benefits of public health.