[Objective] This work aims to investigate differential expression proteins at different development stages of Volvariella volvacea using isobaric tags for relative and absolute quantification (iTRAQ)-coupled two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) proteomics approach. [Methods] Firstly, the proteins extracted from V. volvacea at different development stages were analyzed by SDS-PAGE. Secondly, the tandem mass spcetrometry data obtained from 2D LC-MS/MS were used to search using MASCOT search engine. After that, principal component analysis, hierarchical clustering and Gene Ontology were used to analyze the detected results. [Results] Results showed that 1 039 protein groups, included a total of 2 335 unique peptides, were identified. Among them, 1 030 protein groups were provided quantitative information. Contrast to mycelia, 64 up-regulated and 150 down-regulated significantly differential proteins were found in fruiting bodies at different development stages of V. volvacea. Bioinformatics analysis revealed that iTRAQ-coupled 2D LC-MS/MS was a unique method for isolating and identifying protein groups of V. volvacea at different development stages. [Conclusion] It is helpful to insight into the molecular mechanism of the fruiting body formation and development of V. volvacea and other macro-basidiomycetes in the future.