Abstract: In order to expand the sources of Pleurotus ostreatus culture substrates and reduce the production cost, we analyzed the nutritional components of the cotton stalk powder, selected and identified the best fineness of pulverization, optimum formula and optimum variety. Then a complete set of techniques of Pleurotus ostreatus cultivation with cotton stalk powder was formed successfully. So the best fineness of pulverization was 14 mm, the optimum formula was cotton seed hull 25.5%, cotton stalk powder 59.5%, bran 10%, cotton seed meal 4%, cottonseed oil 1%, added 2% lime, and the ratio of material to water was 1:1.7. The optimum Pleurotus ostreatus variety was ‘Suyan NO.1’. With large-area experiment and demonstration, the yield of Pleurotus ostreatus was the same as that cultivated with the cotton seed hull culture material, and the benefits were higher than that cultivated with cotton seed hull culture material.