Microfluidic technology, commonly referred to as lab-on-a-chip technology, represents a state-of-the-art research domain. It primarily centers on the manipulation and precise control of minute volumes of fluids within micrometer-sized channels. This innovative field has captured substantial attention on account of its distinctive characteristics. These features, such as miniaturization, integration, high precision, automation, and remarkable versatility, endow it with the status of a potent instrument for fluid manipulation and sample analysis across a wide spectrum of applications. These applications span multiple disciplines, including biomedical engineering, chemistry, materials science, and environmental sciences. The Special Issue under discussion is dedicated to the application of microfluidic technology in bioengineering. It encompasses a collection of 13 papers, comprising one Editorial, nine Research Articles, and three Reviews. These papers comprehensively cover the latest advancements in microfluidics, including single-cell manipulation and detection, liquid biopsy, molecular diagnosis, interface design, and simulation modeling. The contributions to this Special Issue come from researchers affiliated with 26 institutions in China, the United States, and Germany.