The invention discloses a differential proteomic analysis method using transcriptome sequencing and based on combination of iTRAQ and LC-MALDI. The method comprises the steps: (1) transcriptome sequencing is performed; (2) differential proteomic analysis is used for extracting the total protein fluid of livers of normal group rats and liver cancer group rats, the two groups are mixed respectively,proteins are precipitated and quantified by an acetone precipitation method, each group contains about 100 [mu]g of the total proteins, trypsin enzymolysis, reduction alkylation, iTRAQ labeling and the like are performed successively, and the process is performed by strictly following instructions of an iTRAQ kit; and (3) bioinformatics analysis is performed. A clinical sample is adopted for positioning and quantitative expression verification, an evidence of the clinical sample with clinical relevance is searched, clinical value is evaluated, and a new clue is provided for research of livercancer pathogenesis and a liver cancer mechanism. Liver cancer key molecules screened by the project can lay the research basis for exploration of liver cancer markers related to early detection, classification and prognosis evaluation, and selection of more effective and accurate liver cancer treatment target positions.