Abstract Rapid and sensitive detection of mycotoxins in foodstuffs is of great significance. As a powerful detection tool, biosensing technologies and microfluidic devices have shown a great potential in rapid and on-site detection of mycotoxins. This review comprehensively summarized the latest advances on the construction of microfluidic biosensors and their promising applications in on-site detection of mycotoxins. Finally, future challenges and chances in this significant and promising field were proposed.