Abstract: In order to compare the difference between GC/MS and GC×GC/TOFMS qualitatively and quantitatively, the effects of column sets and operation conditions, such as modulation frequency, column pressure and initial temperature, on two-dimensional separation were investigated, and a method of analyzing semi-volatile neutral constituents in tobacco by GC×GC/TOFMS was established. The semi-volatile neutral constituents in Yunyan 85 (C3F) were analyzed with the two compared methods, the results showed that: 1) more than 400 peaks were separated and 73 constituents were identified by GC/MS, while near 10000 peaks were separated and 569 constituents were identified by GC×GC/TOFMS; 2) the pretty well profiled peak of β-damascenone in GC was seperated into 6 peaks in GC×GC consisting of 1-ethenyloxy-decane, 1-methyl-4-(2-methyloxiranyl) -7-oxabicyclo-4, 1, 0-heptane, biphenyl, β-damascenone, 10-acetylmethyl-3-carene and tetradecane. Therefore, GC×GC/TOFMS is more powerful and more reliable, and more suitable to complex matrix analysis.