The present invention relates to a method for preparing, from wood-based biomass, a high concentration of fermentable sugar which can be effectively used in culturing various industrial fermented bacteria. According to the method of the present invention, biomass can be extracted by hot water prior to a pre-treatment so as to remove extractible substances such as mineral salts to thus minimize the content of impurities in raw materials for an enzymatic saccharification. The biomass from which substances extractible by hot water are removed is pre-treated in the condition where xylan yield rate is maximized, thus achieving maximum inhibition of the generation of over-decomposed products of sugar. Subsequently, fermentable sugar for culturing various industrial fermented bacteria can be prepared in an inexpensive manner by only concentrating, using a separator membrane, the sugar solution obtained by an enzymatic saccharification of the pre-treated solid content obtained by a solid-liquid separation without washing the solid content with water.