Nicotiana rustica L. (NRL) is a type of tobacco plant, and its stalk waste is a potential lignocellulosic source for obtaining cellulose fibers freely available in nature. However, they are left in fields after harvesting, and this study provides a green and sustainable method to reuse tobacco waste. Fiber was obtained by retting the plant stalks in water and decomposing them naturally in three weeks. NRL fiber was characterized by comparing it with known bast fibers, and tests were applied to examine its physical, chemical, mechanical, morphological, and thermal properties. With its high cellulose content (56.6 wt%), NRL fiber had a high tensile strength (113.4 MPa) and a good crystallinity index (70%) that helped it to bond with other fibers in the composite matrix. Furthermore, the fiber is an environmentally friendly alternative to synthetic fibers with a diameter of 36.88 μm and low density (1.5 g/cm3). The NRL fiber was found to have a semi-crystalline structure and large crystalline size, which makes it hydrophobic. The thermal gravimetric analysis showed that it can be durable (353.9 °C) in higher temperatures than the polymerization temperature. As a result, it can be concluded that NRL fiber has the potential to be used as a reinforcement in polymer composites, technical textiles, and agricultural applications.