The G-quadruplex is a noncanonical secondary structure formed by guanine-rich nucleic acid sequences stabilized by metal ions. Multiple types of human health related target detection strategies have been developed based on the G-quadruplex motif, owing to its high structure stability and manipulability. These G-quadruplex DNA based biosensors exhibit high specificity and sensitivity in a variety of detection strategies. In recent years, microfluidic-based detection platforms are pushing the development of molecular detection forward due to their advantages of high-throughput, high integration, low reagent and time consumption, and portability. By integrating the strengths of both G-quadruplex DNA and microfluidics, G-quadruplex-based microfluidic biosensors are emerging as one of the cutting-edge research approaches. This review provides an overview of the detection strategies of G-quadruplex DNA-based biosensors and the classification of microfluidic technologies. It then focuses on the application of G-quadruplex-based microfluidics in the detection of human health-related ions and biomarkers, as well as its innovative applications as drug screening tools. At the end of this review, we discuss the advantages and current challenges of G-quadruplex-based microfluidic detection platforms.