In order to determine protein in tobacco fast and accurately, the classical method for determining protein in tobacco was modified in three aspects: 1) water soluble nitrogen in tobacco was removed by ultrasonic extraction with acetic acid; 2) the tobacco sample was digested in hydrogen peroxide and sulphuric acid mixture solution (2/1, v/v) by heating for ten minutes, while the digesting time of the classical method, Kjeldahl method, for nitrogen determination was over 2.5 hours; 3) the digested sample solution were determined with the colorimetry by using salicylic acid as a color. The results showed that this method was fast and simple, the average recovery was 101.1%, RSD was below 2%, and it could be used in fast determination of protein in tobacco.