Abstract: In order to explore the relationship between the yield of Agaricus bisporus with physicochemical properties of the secondary fermentation substrates used for Agaricus bisporus cultivation and the effect of various factors on the yield formation, to provide a scientific reference for the production and evaluation of fermentation substrates increasing the yield of Agaricus bisporus, 10 batches of different raw materials were used to make compost and tunnel fermentation technology was used for fermentation. The temperature and humidity of the mushroom house were monitored and adjusted through the air conditioning automatic control system to ensure the growth of Agaricus bisporus. The fermented substrates were sampled , and the physicochemical properties and enzyme activity of which were determined, such as water content, pH, EC, carbon content, nitrogen content, carbon-nitrogen ratio, cellulose and hemicellulose content and the enzyme activity of xylanase, carboxymethyl cellulase (CMC), α-cellobiohydrolase, xylosidase, α-arabinofuranosidase and so on, totally 13 factors. Principal component analysis and cluster analysis were used to classify these factors. Counting the yield of each batch of Agaricus bisporus and using multiple regression analysis the relationship between the factors and the yield of Agaricus bisporus were established. The results showed that hemicellulase and cellulase, pH, EC, nitrogen content and carbohydrate related factors were the main components of the fermentation substrates. Principal component analysis extracted 4 principal components from 13 factors, which were divided into carbohydrate change factor, main degrading enzymes of cellulose, EC value and nitrogen content, main degrading enzymes of hemicellulose. The variance contribution rate were 31.18%, 18.45%, 18.34%, and 16.08%, respectively. Their cumulative contribution rate was 84.04%, which represented most of the original factor. Cluster analysis clustered 13 factors into 4 categories, namely carbohydrate change factor, EC value and nitrogen content, degrading enzymes of hemicellulose, and pH value. Cluster analysis is simple and intuitive but can only classifies similar factors into one class. Principal component analysis can not only extract 4 principal components, but also have a high cumulative contribution rate. Compared with cluster analysis, the results of principal component analysis are more objective and reasonable. The equation between yield of Agaricus bisporus with the carboxymethyl cellulase activity, xylanase activity and cellulose content of secondary fermentation substrates was established by multiple regression analysis ,which fitting well. There were positive linear relationship between xylanase and carboxymethyl cellulase activity and the yield of Agaricus bisporus. The cellulose content had a negative linear relationship with the yield of Agaricus bisporus. This equation can be used to predict the possible yield of Agaricus bisporus when various physicochemical factors and enzyme activity of fermentation substrates are measured. The comprehensive analysis of the three methods showed that the yield of Agaricus bisporus was affected by various factors of fermentation substrates together. The activities of xylanase and carboxymethyl cellulase were more closely related to the yield of Agaricus bisporus than other factors, and positively correlated with the yield of Agaricus bisporus.