Fermentable sugar is the most important component in sweet sorghum stem for its first-generation ethanol production. Estimation of fermentable sugar yield is required for sweet sorghum breeding, production, but related estimation methods available now are not so efficient. In order to establish a new method of estimating fermentable sugar yield, sweet sorghum stems were milled with a small three roll miller to recover carefully the juice and bagasse. The contents of sugars in the juice and water extraction of bagasse were analyzed with high performance liquid chromatography. Contents of soluble matter and sugars (glucose, fructose and sucrose) of the extracted juice and calculated juice of the bagasse (moisture content plus soluble matter content) were compared. The results showed that there is no significant difference in the total fermentable sugar content between squeezed juice and residue juice. So, the following four methods of analysis and estimation for sugars content of the whole stem was studied and compared: (1) analyzing juice only (but needs to determine moisture content of bagasse or stem, (2) analyzing bagasse only, (3) analyzing both juice and bagasse and (4) analyzing dried stems. The results showed that it is feasible to estimate sugars content of the whole stem by analyzing the juice or the bagasse only, and the most simplified method was that analyzing the juice and drying the bagasse. The following three formula was postulated that can be used in the estimation of sugars contents: (1) S-s = (J +B x So(j) x W-b / W-j) x S-j; (2) S-s-(1-B x D(b)xU(b))xS(b)/(W-b + So(b)); (3) S-s-J x S-i + B x S-b, where Ss is sugar content of stem (%), J is juice extraction (%), B is bagasse rate after one milling (%), Soj is content of soluble matter in extracted juice(%), W-b is water content of bagasse (%), W-b is water content of extracted juice (%); S-j is sugar content of juice(%); D-b is content of dried matter in bagasse(%); S-b is sugar content of fresh bagasse; S-ob is content of soluble matter in fresh bagasse. Fermentable sugar yield (FSY, MgHa(-1)) = Green stem yield (GSY, MgHa(-1)) x S-s (%).