Abstract: To match the homology of protein detection reference materials from flue-cured tobacco, proteins with higher mass fractions were prepared from flue-cured tobacco sources, and the extraction and purification processes of water-insoluble tobacco proteins were studied. In addition, a crude extract of water-insoluble proteins from tobacco leaves was prepared on a pilot scale. The effects of four single factors, including pH, mass fraction of sodium chlorite, heating temperature, and addition amount of ammonium sulfate, on the purification effect of crude water-insoluble tobacco proteins were investigated. The mass fractions of water-insoluble tobacco proteins were determined by the Kjeldahl method, and the values of L^*, a^*, b^*, and ΔE^*_ab were measured and calculated using a colorimeter. The results showed that: 1) The optimal purification process was achieved by using 5 grams of crude water-insoluble tobacco proteins, which were reacted with 50 mL of 3% (mass fraction) sodium chlorite solution at pH 8 and 60 ℃ for 30 minutes, and then salted out with 10 grams of ammonium sulfate. 2) Under the optimized processing condition, the mass fraction and yield of water-insoluble tobacco proteins were 88.41% and 12.57%, respectively, and the ΔE^*_ab value between the sample and the control group was the largest.