In this study, the culture requirements of the Cerrena unicolor OBCC 5005 strain were determined to optimize bioactive exopolysaccharide production in submerged culture. The effects of initial medium pH, carbon and nitrogen sources, inoculum age and amount, and mineral source on exopolysaccharide and mycelial biomass production by the C. unicolor OBCC 5005 strain were studied using a one-factor-at-a-time method. The highest exopolysaccharide production was obtained when culture parameters were used as initial medium pH: 5.5, 5% sucrose, 5% mycological peptone, and 5% of 4-day inoculants in the presence of 5 mM Fe2+. Optimized culture conditions at a flask scale were applied to a 3-L stirred tank reactor. As a result, 7.92 g/L and 7.34 g/L maximum exopolysaccharide production in optimized conditions at flask and stirred-tank reactor scales were achieved, respectively. The present study is the first to prove that C. unicolor can yield high bioactive exopolysaccharide production at flask and stirred-tank reactor scales.