Multifunctional Janus particles have a variety of applications in a wide range of fields. However, to achieve many of these applications, high-throughput, low-cost techniques are needed to synthesize these particles with precise control of the various structural/physical/chemical properties. Microfluidics provides a unique platform to fabricate Janus particles using carefully controlled liquid flow in microfluidic channels to form Janus droplets and various types of solidification methods to solidify them into Janus particles. In this Focus article, we summarize the most recent representative works on Janus particle fabrication in microfluidics. The applications of Janus particles in biomedical areas are emphasized. We believe that microfluidics-enabled multifunctional Janus particles could resolve multiple prevalent issues in biomedicine (e.g., disease monitoring at an early stage, high-throughput bioassays, therapeutic delivery) if persistent effort and collaboration are devoted to this direction.