Abstract: In order to objectively evaluate the brittleness of tobacco leaves, a quantitative evaluation method was developed by using the puncture mode of a texture analyzer. The optimized test parameters were determined through single factor and response surface methodology, and the differences of brittleness of tobacco leaf samples with different moisture contents from different planting areas and stalk positions were evaluated by the developed method. The results showed that: 1) The optimized conditions of the texture analyzer for determining tobacco leaf brittleness were based on the P/2N needle probe, the speeds before, during and after test 1.0, 1.5 and 10.0 mm/s respectively, test distance 5 mm, and trigger force 5 g. The intra- and inter-day precisions of this method were less than 4.50% and less than 3.00% respectively. 2) There were some differences in tobacco leaf brittleness among leaves from different planting areas; however, there were significant differences among leaves from different stalk positions in the rank of lower leaf > upper leaf > middle leaf. 3) The moisture content in tobacco leaf significantly affected its brittleness. When the moisture content was less than 20%, tobacco leaf brittleness decreased significantly with the increase of moisture content. 4) With the rise of environmental humidity, the diameter of puncture in tobacco leaf decreased gradually, the section became smooth, and the microcracks were less and not developing.