A microfluidic affinity interface is a substrate modified with recognition molecules in a microfluidic device for specific target capture based on molecular recognition. The utilization of microfluidic affinity interfaces holds great significance in microfluidic applications, such as analytical detection, liquid biopsy, and single-cell analysis. This review focuses on the regulation of molecular recognition on a microfluidic affinity interface. Based on the theoretical mode of target capture, we discuss strategies to improve the molecular recognition efficiency from two aspects: enhancement of encounter rate between target and affinity interface, and improvement of binding kinetics and thermodynamics on the microfluidic affinity interface. Finally, some perspectives relating to future research directions and challenges are discussed.