Abstract: The strain Klebsiella oxytoca THLC0409 was isolated from a lignocellulose-degrading microflora and used in the direct conversion of Napiergrass powder to ethanol. Central Composite Design (CCD), part of the Response Surface Methodology (RSM) was adopted to study the effects of the fermentation time, the initial pH of the liquid medium, the cultivation temperature, and the yeast extract (YE) concentration on ethanol production. A second-order model that relates the ethanol concentration to four basic factors was developed. Regression analysis revealed that the maximum ethanol concentration of 472 ppm could be reached at a fermentation time of about 11 days, an initial pH of 7.04, a cultivating temperature of 31 °C, and a YE concentration of 7.0 g l−1. The adjusted R-square of the regression model was 0.996, indicating a good fit at 99.6% confidence level. The highest ethanol yield and productivity were estimated to be 82 g kg−1 and 360 mg kg−1 h−1, respectively.