Microfluidic biosensors integrating fluid control, target recognition, as well as signal transduction and output, have been widely used in the field of disease diagnosis, drug screening, food safety and environmental monitoring in the past two decades. As the central part and technical characteristics of microfluidic biosensors, the fluid control is not only associated with accuracy and convenience of the sensors, but also affects the material selection and working mode of the sensors. This review summarizes the fluid driving forces for microfluidic biosensors, including gravity, capillary force, centrifugal force, pressure, light, sound, electrical, and magnetic forces. Then, the recent advances in microfluidic biosensors for the detection of viruses, cells, nucleic acids, proteins and small molecules are discussed. Finally, we propose the current challenges and future perspectives of microfluidic biosensors. We hope this review can provide readers with a new perspective to understand the technical characteristics and application potential of microfluidic biosensors.