Insomnia is an economic burden and public health problem. This study aimed to explore potential biological pathways and protein networks for insomnia characterized by wakefulness after sleep. Proteomics analysis was performed in the insomnia group with wakefulness and the control group. The differentially expressed proteins (DEPs) were enriched, then hub proteins were identified by protein-protein interaction (PPI) network and verified by parallel reaction monitoring (PRM). Compared with the control group, the sleep time and efficiency of insomnia patients were decreased, awakening time and numbers after sleep onset were significantly increased (P < 0.001). The results of proteomic sequencing found 68 DEPs in serum under 1.2-fold changed standard. These DEPs were significantly enriched in humoral immune response, complement and coagulation cascades, cholesterol metabolism. Through PPI network, we identified 10 proteins with the highest connectivity as hub proteins. Among them, differential expression of 9 proteins was verified by PRM.We identified the hub proteins and molecular mechanisms of insomnia patients characterized by wakefulness after sleep. It provided potential molecular targets for the clinical diagnosis and treatment of these patients, and indicated the immune and metabolic systems may be closely related to insomnia characterized by wakefulness after sleep.