Abstract: Tobacco is the raw material of cigarettes in general, but its leaf is one of the most abundant protein resources in the world. The proteins in tobacco leaves can be divided into water-soluble and water-insoluble proteins. Half of the soluble proteins are FI proteins (Fraction I proteins), and the other half are FII proteins (Fraction II proteins). With the development of technology, many new technologies have been applied to protein separation, such as aqueous two-phase extraction, ultrafiltration, chromatography, and electrophoresis. According to the relevant research literatures at home and abroad, several commonly used extraction techniques and the latest progress for separating tobacco leaf proteins were reviewed and discussed in this article, and their application prospects were described.