During the two-stage submerged fermentation of medicinal mushroom Cordyceps militaris, it was found that K+, Ca2+, Mg2+, and Mn2+ were favorable to the mycelial growth. The EPS production reached the highest levels in the media containing Mg2+ and Mn2+. However, Ca2+ and K+ almost failed to increase significantly exopolysaccharides (EPS) production. Sodium dodecyl sulfate (SDS) significantly enhanced EPS production compared with that of without adding SDS when SDS was added on static culture stage of two-stage cultivation process. The presence of Tween 80 in the medium not only simulated mycelial growth but also increased EPS production. By response surface methods (RSM), EPS production reached its peak value of 3.28 g/L under optimal combination of 27.6 mM Mg2+, 11.1 mM Mn2+, and 0.05 mM SDS, which was 3.76-fold compared with that of without metal ion and surfactant. The results obtained were useful in better understanding the regulation for efficient production of EPS of C. militaris in the two-stage submerged culture.