Provided is a formula of an oyster mushroom culture medium. The oyster mushroom culture medium is prepared from, by weight, 85-90% of main materials and 10-15% of auxiliary materials. The main materials include cottonseed hulls, corncobs, corn straw, corn meal and wood dust. The auxiliary materials include wheat bran, quick lime, microelement mixture, glucose and sterilizing powder. A preparing method of the oyster mushroom culture medium includes the steps of S100, washing and airing the main materials respectively; S200, conducting smashing, mixing and stirring to obtain a main material mixture; S300, spraying water to the main material mixture; S400, adding the auxiliary materials to the main material mixture, adding gibberellin, spraying water to be stirred, and standing for 10-20 minutes; S500, filling a plastic bag with the mixture obtained in the step S400, and conducting high-temperature vapor sterilizing. The culture medium contains various nutritional substances, antibacterial and sterilizing quick lime, microelements and mineral substances, and the growth of oyster mushrooms is promoted.