The optimisation of submerged culture conditions and nutritional requirements was studied for the production of exopolysaccharide (EPS) fromPleurotus nebrodensis. The optimal temperature and initial pH for both mycelial growth and EPS production in shake flask cultures were 25 °C and 8.0, respectively. Maltose was found the most suitable carbon source for both mycelial biomass and EPS production. Yeast extract was favourable nitrogen source for both mycelial biomass and EPS production. Optimum concentration of each medium component was determined using the orthogonal matrix method. The optimal combination of the media constituents for mycelial growth and EPS production was as follows: 200 g l−1 bran, 25 g l−1 maltose, 3 g l−1 yeast extract, 1 g l−1 KH2PO4, 1 g l−1 MgSO4 7H2O. Under the optimal conditions, the mycelial biomass (4.13 g l−1) and EPS content (2.40 g l−1) ofPleurotus nebrodensis was 2.3 and 3.6 times compared to the control with basal medium respectively.