Cancer is a major cause of disease-related deaths worldwide, and early diagnosis involving detecting biomarkers of tumors can improve the cure rate and prognosis of patients. Biomarkers are signature proteins that can distinguish diseased cells from healthy cells, facilitating the diagnosis and treatment of diseases, especially cancer. Aptamers are single-stranded oligonucleotides that can recognize target proteins with high affinity and specificity. The development of biomarkers identified by aptamers has experienced extensive progress in current applications. The combinatorial strategy of cell-SELEX technology and proteomics analysis makes targeted protein identification more cost-effective and efficient and improves the success rate of discovery of multiple biomarkers simultaneously. In this methodology, biomarkers are identified via a series of operations such as screening of aptamers, separation, extraction, and analysis of target proteins, which has brought about the discovery of a number of new biomarkers of cancer. This review summarizes the current strategies, challenges, and potential applications for biomarker discovery using aptamers engendered by cell-SELEX.