Cardiovascular disease is currently the leading cause of death worldwide. Atherosclerosis is an important pathological basis of cardiovascular disease, and its early diagnosis is of great significance. Urine is more conducive in the accumulation and response of changes in the physiological state of the body and is not regulated by homeostasis mechanisms, so it is a good source of biomarkers in the early stage of disease. In this study, ApoE-/- mice induced by a high-fat diet for 5 months were used to construct an animal model of atherosclerosis. Urine samples from the experimental group and control group, which were C57BL/6 mice fed a normal diet, were collected at seven time points. Proteomic analysis was used for internalcontrol and intergroup control. Internal control results showed a significant difference in the urinary proteome before and after a 1-week high-fat diet, and several differential proteins have been reported to be associated with atherosclerosis or for use as biomarkers. The results of the intergroup control indicated that the biological process enriched by the GO analysis of the differential proteins corresponded to disease progression. Differences in chemical modifications of urinary proteins have also been reported to be associated with the disease. This study demonstrates that urinary proteomics has the potential to monitor changes in the body sensitively and provides the possibility of identifying early biomarkers of atherosclerosis.