Aptamers are single-stranded DNA or RNA oligomers, identified from a random sequence pool, with the ability to form unique and versatile tertiary structures that bind to cognate molecules with superior specificity. Their small size, excellent chemical stability and low immunogenicity enable them to rival antibodies in cancer imaging and therapy applications. Their facile chemical synthesis, versatility in structural design and engineering, and the ability for site-specific modifications with functional moieties make aptamers excellent recognition motifs for cancer biomarker discovery and detection. Moreover, aptamers can be selected or engineered to regulate cancer protein functions, as well as to guide anti-cancer drug design or screening. This review summarizes their applications in cancer, including cancer biomarker discovery and detection, cancer imaging, cancer therapy, and anti-cancer drug discovery. Although relevant applications are relatively new, the significant progress achieved has demonstrated that aptamers can be promising players in cancer research.