The active ingredients from tobacco extracts were continuously separated and purified using a homemade free-flow electrophoresis apparatus. A rectangular free flow electrophoresis device was constructed for the continuous separation and preparation, and the operating conditions of the device were optimized. The fractions obtained from the free-flowing component collection unit were then detected by HPLC and GC-MS. The results showed that a 90% methanol-water solution could maximize the extraction of the active components from tobacco. Chlorogenic acid and nicotine were enriched in three and four of 24 fractions, respectively, after free-flow isoelectric focusing electrophoresis. 2-Hydroxy-2-cyclopentene-1-one, 1-(2-methyl-1,3-oxathiolan-2-yl) ethanone, nornicotine, cotinine, and scopolamine were separated and enriched synchronously. Overall, the use of free-flow electrophoresis technology for the separation and purification of the active substances in tobacco can improve the comprehensive utilization rate of tobacco.