The invention discloses a method used for discovering and identifying liver cancer serum differential expression proteins and verifying marker proteins. According to the method, iTRAQ labeling MALDI-TOF MS/MS technology is adopted for discovering and identifying liver cancer serum differential expression proteins and MRM verifying of marker proteins. According to the method, searching on effectivetumor markers possesses important meaning on canceration mechanism study, disease early stage diagnosis, and prognosis. Selected novel liver cancer protein markers are subjected to in vivo and in vitro experiments such as gene knockin and knockout, and nude mice transplanted cancer, influences of blocking or up-regulating of expression of candidate genes on tumor biological behavior are observed,and key proteins capable of influencing human liver cancer occurrence and development are positioned, the effects of the key proteins in a system signal network is analyzed based on bioinformatics analysis, and the feasibility, the liver cancer diagnosis value, and the clinical significance of the key proteins in clinical applications including liver cancer prevention, early stage diagnosis, andprognosis is evaluated based on MRM absolute quantitative verification.