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Due to excellent characteristics, there is a vast planting area of eucalyptus in China. Its cultivation, production and processing processes will produce a large number of eucalyptus bark and sawdust, of which surplus resources are rich. It has been reported to cultivate edible fungi using eucalyptus sawdust. To analyze the difference of metabolites and their pathways in Pleurotus pulmonarius cultivated with three different substrates among eucalyptus bark, eucalyptus sawdust and miscellaneous sawdust. Based on the Ultra-High Performance Liquid Chromatography tandem Mass Spectrometry (UHPLC-MS) technique, an untargeted metabolomic study was performed on the differences metabolites of fruit bodies of Pleurotus pulmonarius under different cultivation substrates by and Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA). The results were as follows: (1) The screening results based on Value Importance in Projection (VIP) >1 and T test P0.05). In conclusion, using the by-products of eucalyptus processing (eucalyptus bark and eucalyptus sawdust) as the main cultivation substrates of P. pulmonarius, especially eucalyptus sawdust, can realize resource recycling, reduce production costs and improve economic benefits. This study provides a theoretical basis for the application of eucalyptus processing by-products in the cultivation of edible fungi.