Due to its high resolution, high throughput, and low cost, nanoimprint lithography (NIL) and its variations are considered the most promising lithographic techniques for high-volume manufacturing of nanoscale devices. Compared to nanostructures fabricated by chemical synthesis and self-assembly, those fabricated by nanolithography and thin-film technology offer precise control of the structures’ shape, size, and positioning on a substrate. In this article we will focus on two application areas of nanofabrication using NIL. The first one is in the field of biosensing and covers nanostructured plasmonic biosensors, nonplasmonic biosensors such as those based on photonic crystals, electrical or electrochemical biosensors, and biosensors based on nanoelectromechanical systems. The majority of biosensors detect the biobinding events including DNA hybridization and antibody–antigen binding; with the remainder detect a fingerprint of target biomolecules or a labeling molecule or an attached particle. Fabrication of micro- and nano-fluidics using NIL for biomedical applications will also be introduced briefly. The second application area is in the field of tissue engineering and covers mainly contact guidance of cells cultured on micro- or nanostructured substrates.