The hydrolyzate of banana peels containing abundant fermentable sugars as glucose, xylose, mannose, and arabinose was successfully used as a novel substrate for the efficient production of glutathione by Candida utilis SZU 07-01. Xylose was first selected as the sole carbon source for glutathione production, medium optimization for better cell growth and higher glutathione using response surface methodology consisting of PB design, the steepest ascent experiment and CCD was carried out, and the optimal combination of nutrients was obtained as follows: xylose 20 g/L, (NH4)2SO4 9.59 g/L, KH2PO4 3 g/L, L-methionine 5.72 g/L and MgSO4 0.20 g/L. The maximum dry cell weight and glutathione achieved using the optimized medium were 7.36 g/L and 154.32 mg/L, respectively. Following with the content in this medium, other sugars like glucose, mannose and arabinose were chosen as the sole carbon source and all tested available for glutathione production. Based on these results, the hydrolyzate of banana peels was selected as a novel substrate, and a high DCW of 7.68 g/L and glutathione yield of 111.33 mg/L were obtained with the initial sugar concentration of 20 g/L in the hydrolyzate of banana peels.