Phage display is a vital tool for the discovery and development of affinity reagents such as antibodies and peptides, which have great potential in imaging, molecular recognition, biosensors, targeted delivery and other clinical applications. However, affinity reagents obtained by phage display are often subjected to a process called biopanning, which is considered time-consuming, labor-intensive and lacks accurate control, limiting the acquisition of high-quality affinity reagents. Over the last two decades, several microfluidic approaches have been designed to simplify the conventional biopanning process and to realize precise control. To better understand the advantages of microfluidics over traditional biopanning and the potential of microfluidics for other molecular screening strategies, we provided an overview of recent applications of microfluidics in phage display. Additionally, the next challenges and outlooks are discussed.