Abstract: Tobacco has important economic value and strong potential for synthetic biology applications. With its large biomass and active secondary metabolism, it serves not only as a raw material for the traditional cigarette industry but also as an ideal candidate for the development of plant bioreactors. However, nicotine, the primary alkaloid, provides flavor and addictiveness but also poses industrial challenges. Over the past three decades, global harm-reduction policies have pushed the industry to lower nicotine levels to protect public health, prompting a critical transformation. High nicotine content also limits tobacco's use as a chassis in biomanufacturing, creating demand for low- or zero-nicotine materials. Developing low-nicotine tobacco is now a priority. This review summarizes recent advances in low-nicotine tobacco research, including progress in nicotine biosynthesis, degradation, and transport, and describes the regulatory network of nicotine metabolism from three perspectives: genetic regulation (core transcription factors from NIC1/NIC2 loci), phytohormone signaling (crosstalk among jasmonic acid, ethylene, and auxin), and environmental responses (light, temperature, water, and nitrogen). It also reviews production technologies for low-nicotine tobacco, such as traditional breeding, genetic engineering, agronomic practices, chemical regulation, and microbial degradation. Technical challenges and future directions are discussed, including network analysis and precision breeding with multi-omics, the development of synthetic biology technology, and the exploration of industrialization strategies. This review provides a system reference for the genetic improvement and industrial application of low-nicotine tobacco.