N-acetylglucosamine (GlcNAc) is a crucial structural component of biologically important polysaccharides across species, such as hyaluronic acid in humans, chitin in animals, and peptidoglycan in bacteria. As a reducing functional monosaccharide, GlcNAc exhibits multiple bioactivities, including antioxidant, anti-aging, antimicrobial, and immunomodulatory properties. Recent years have witnessed significant progress in GlcNAc production via chemical hydrolysis, enzymatic hydrolysis, and microbial biosynthesis. Notably, microbial biosynthesis technology has garnered significant attention due to its environmentally friendly, cost-effective, and efficient advantages. Currently, this approach has initially enabled the efficient biosynthesis of GlcNAc through the metabolic regulation and optimization of various microbial chassis. As preparation technologies advance and production efficiencies improve, the applications of GlcNAc in food fields are expanding rapidly. This article provides a comprehensive review of the principal methods for GlcNAc production, with a focus on the latest advancements in microbial synthesis. In addition, it explores the multifunctional applications of GlcNAc in food material development, nutritional function optimization, preservation and quality control. Furthermore, by integrating recent breakthroughs in biotechnology, coupled with innovation strategies driven by artificial intelligence, the sustainable production and future applications of GlcNAc are prospected, aiming to offer a scientific foundation for its industrial development and large-scale commercialization.