As a booming engineering technology, the microfluidic chip has been widely applied for replicating the complexity of human intestinal micro-physiological ecosystems . Biosensors, 3D imaging, and multi-omics have been applied to engineer more sophisticated intestinal barrier-on-chip platforms, allowing the improved monitoring of physiological processes and enhancing chip performance. In this review, we report cutting-edge advances in the microfluidic techniques applied for the establishment and evaluation of intestinal barrier platforms. We discuss different design principles and microfabrication strategies for the establishment of microfluidic gut barrier models . Further, we comprehensively cover the complex cell types (e.g., epithelium, intestinal organoids, endothelium, microbes, and immune cells) and controllable extracellular microenvironment parameters (e.g., oxygen gradient, peristalsis, bioflow, and gut-organ axis) used to recapitulate the main structural and functional complexity of gut barriers. We also present the current multidisciplinary technologies and indicators used for evaluating the morphological structure and barrier integrity of established gut barrier models . Finally, we highlight the challenges and future perspectives for accelerating the broader applications of these platforms in disease simulation, drug development, and personalized medicine. Hence, this review provides a comprehensive guide for the development and evaluation of microfluidic-based gut barrier platforms.