GLYCEROL is considered as a great tool for industrial applications. Mechanism of glycerol production by yeast cell has become highly attractive and interesting. Many factors affect glycerol yield throughout fermentation process. For this reason, an osmotolerant glycerol producer yeast strain Wickerhamomyces anomalus AUMC 11687 was selected for optimization of glycerol production conditions by using response surface methodology. Four independent variables (temperature, pH, sugar and glycerol concentration as osmoregulatory) were studied using the Box-Behnken Design methodology with 4-factors and 3-levels. The best conditions were calculated by statistical regression analysis and surface plots. The optimal production parameters for maximum glycerol production were at 33.34ºC, 6.9 pH, 22.454% sugar concentration and 6.307% initial glycerol as osmoregulator. The concentration of glycerol and residual sugars under these optimal conditions were 129.65 and 7.404gL-1, respectively. Results recorded during this study showed that the osmotolerant W. anomalus AUMC 11687 is a promising yeast with great potentials for application in glycerol production on large scale for industrial purposes.