Isoporous membranes are versatile structures with numerous potential and realized applications in various fields of science such as micro/nanofiltration, cell separation and harvesting, controlled drug delivery, optics, gas separation, and chromatography. Recent advances in micro/nanofabrication techniques and material synthesis provide novel methods toward controlling the detailed microstructure of membrane materials, allowing fabrication of membranes with well-defined pore size and shape. This review summarizes the current state-of-the-art for isoporous membrane fabrication using different techniques, including microfabrication, anodization, and advanced material synthesis. Various applications of isoporous membranes, such as protein filtration, pathogen isolation, cell harvesting, biosensing, and drug delivery, are also presented.