In order to explore the changes in protein expression of large yellow croaker under high temperature stress, tandem mass tag（TMT）combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to perform proteome analysis on the liver tissues between the high temperature tolerance group and the high temperature sensitive group of large yellow croaker. A total of 3 369 proteins were identified, of which 687 were differentially expressed proteins, including 281 up-regulated proteins and 406 down-regulated proteins. 13 randomly selected proteins were validated with parallel reaction monitoring (PRM) assays, which showed that they were consistent with the TMT-labeled proteome analysis results. Subsequently，through bioinformatics analysis and found that high temperature stress has a significant impact on the protein folding and energy metabolism of large yellow croaker. Among them, heat shock proteins 70, calreticulin, glucose-regulated protein and pyruvate kinase play an important role in the response to high temperature stress, This study is helpful to further analyze the high temperature adaptation mechanism of large yellow croaker, and provides reference for breeding high temperature resistant species of large yellow croaker.