The enzyme assisted extraction conditions of polysaccharide from Cordyceps militaris mycelia were firstly investigated by kinetics analysis and the optimal operating was found to be: extraction temperature 40 °C; solid-solvent ratio 1:20; extraction pH 4.0; cellulase concentration 2.0%. The polysaccharide extraction yield was 5.99% under these optimized conditions. Furthermore, a fundamental investigation of the biosorption of Pb2+ from aqueous solution by the C. militaris polysaccharide was performed under batch conditions. The suitable pH (5.0), polysaccharide concentration (0.20 g L−1), initial Pb2+ concentration (300 mg L−1) and contact time (40 min) were outlined to enhance Pb2+ biosorption from aqueous medium. The Langmuir isotherm model and pseudo first order kinetic model fitted well to the data of Pb2+ biosorption, suggesting the biosorption of Pb2+ onto C. militaris polysaccharide was monolayer biosorption and physical adsorption might be the rate-limiting step that controlled the adsorption process. FTIR analysis showed that the main functional groups of C. militaris polysaccharide involved in adsorption process were carbonyl, carboxyl, and hydroxyl groups.