The pretreatment conditions，including extraction solvent，derivatization time and temperature，as well as internal standard were optimized for metabolome analysis of Botrytis cinerea(B-cinerea)，an important pathogenic fungus that could induce a severe grey mold on plants.The results showed that，using salicin as the internal standard，a mixture of methanol and water(80∶20) with a dosage of 4.0 mL for 0.1 g of mycelium as extraction reagent，a silylation of N，O-bis(trimethylsilyl)trifluoroacetamide with methoxylamine for 6 h of derivation at 37 ℃ was the optimal for most kinds of metabolites in derivatives yield and stability.In B-cinerea hypha metabolome，210 peaks were detected by GC-MS， and the metabolome was identified as a group of metabolites including mainly amino acids，alcohols，organic acids，sugars and others.Among the metabolites，50 of them were identified by NIST 2008 with a match quality of 80% or more, and part of them by standards.As a result，typical 17 metabolites obtained good linearities in the range of 1.0-100 μg/mL with correlation coefficients more than 0.98.The limits of detection and limits of quantitation were in the range of 0.02-10.0 ng/mL and 0.1-34.0 ng/mL，respectively.Investigation on the reproducibility of the optimal method suggested that the RSD for 89.05% metabolites detected were in the range of 0.01%-16.8%.The good reproducibility implied that the method established was suitable for the metabolome detection of mycelial fungi,and could provide a good reference for microorganism metabolomics research in the field of agricultural and medical science.