Over the past decades, microfluidic devices based on many advanced techniques have aroused widespread attention in the fields of chemical, biological, and analytical applications. Integration of microdevices with a variety of chip designs will facilitate promising functionality. Notably, the combination of microfluidics with functional nanomaterials may provide creative ideas to achieve rapid and sensitive detection of various biospecies. In this review, focused on the microfluids and microdevices in terms of their fabrication, integration, and functions, we summarize the up-to-date developments in microfluidics-based analysis of biospecies, where biomarkers, small molecules, cells, and pathogens as representative biospecies have been explored in-depth. The promising applications of microfluidic biosensors including clinical diagnosis, food safety control, and environmental monitoring are also discussed. This review aims to highlight the importance of microfluidics-based biosensors in achieving high throughput, highly sensitive, and low-cost analysis and to promote microfluidics toward a wider range of applications.