This study aimed to investigate the enzymatic hydrolysis of mannitol using Viscozyme$^{(R)}$ L, Celluclast$^{(R)}$ 1.5 L, Saczyme$^{(R)}$, Novozym$^{(R)}$, Fungamyl$^{(R)}$ 800 L, Driselase$^{(R)}$ Basidiomycetes sp., and Alginate Lyase, and to optimize of reaction conditions for production of reducing sugar. Response surface methodology (RSM) based on central composite rotatable design was used to study effects of the independent variables such as enzyme (1-9% v/w), reaction time (10-30 h), pH (3.0-7.0) and reaction temperature ($30-70^{circ}C$) on production of reducing sugar from mannitol. The coefficient of determination ($R^2$) of $Y_1$ (yield of reducing sugar by Viscozyme$^{(R)}$ L) and $Y_3$ (yield of reducing sugar by Saczyme$^{(R)}$) for the dependent variable regression equation was analyzed as 0.985 and 0.814. And the p-value of $Y_1$ and $Y_3$ showing 0.000 and 0.001 within 1% (p < 0.01), respectively, was very significant. The optimum conditions for production of reducing sugar with Viscozyme$^{(R)}$ L were 9.0 % (v/w) amount of enzyme, 30.0 hours of reaction time, pH 4.5 and $30.0^{circ}C$ of reaction temperature, and those with Saczyme$^{(R)}$ were 9.0% (v/w) of amount of enzyme dosage, 30.0 h of reaction time, pH 7.0 and $30.0^{circ}C$ of reaction temperature, consequently, the predicted reducing sugar yields were 22.5 and 27.9 mg/g-mannitol, respectively.