In this study, the production of polysaccharides by kefir grains during milk fermentation was optimized with respect to the amount of nutrients added to the fermentation medium (sterilized milk) employing one-factor-at-a-time and orthogonal array design methods. The results showed that the optimal carbon sources added to sterilized milk were 3% maltose, 4% sucrose and 4% glucose, resulting in a maximum yield of polysaccharides of 1.92 g/L, the optimal nitrogen sources were 0.2% yeast extract, 1% peptone and 0.004% L-tyrosine, resulting in a maximum yield of polysaccharides of 2.07 g/L, and the optimal inorganic salts were 0.4% KH2PO4, 0.08% MgSO4·7H2O, 0.02% CaCO3 and 0.25% NaCl, resulting in a maximum yield of polysaccharides of 2.01 g/L.