Abstract: 【Objective】 To provide theoretical basis and technical support for the investigation, isolation and research of nematode disease in Agaricus bisporus, through determining the scientific method of isolating nematodes from Agaricus bisporus culture medium. 【Methods】 Nematoda was isolated from Agaricus bisporus substrate by Beman funnel method. The effects of different substrate types, different production methods and media treatment methods on the occurrence of Nematoda were evaluated. 【Results】 The results showed that there were significant differences in nematode isolation rate among different matrix materials, the separation rates of cotton stalk, wheat straw, cottonseed hull and soil was 86.05%, 70.91%, 37.98% and 28.03%, respectively; The porosity of wheat straw, cotton stalk, cottonseed hull and soil was 3.36, 2.48, 2.02 and 1.24 mL/g, respectively; There were significant differences in nematode concentration under different production methods and treatments; The nematode concentration in the substrate was the lowest under the conditions of industrial production and secondary fermentation, the nematode concentration was 0 / 100g and 59.21 / 100g respectively in the culture medium, and was 12 / 100g and 79 / 100g in soil covering materials respectively, before and after fruiting; The nematode concentration in the substrate was the highest under the conditions of agricultural production and one-time fermentation, the nematode concentration was 1,556.33/100g and 5,371.33/100g respectively in the culture medium, and was 79/100g and 5,236.67/100g in soil covering materials respectively, before and after fruiting. 【Conclusion】 There was a significant difference in the rate of nematode isolation among different matrix materials, and there was a significant positive correlation between the rate of nematode isolation and porosity. The number of nematodes is closely related to the mode of production and the method of culture material treatment, the more advanced the mode of production and the more thorough the fermentation treatment of culture material, the lower the number of nematodes.