Extrachromosomal DNA (ecDNA) is circular DNA that plays an important role in the development and heterogeneity of cancer. The rapid evolution of methods to detect ecDNA, including microscopic and sequencing approaches, has greatly enhanced our knowledge of the role of ecDNA in cancer development and evolution. Here, we review the molecular characteristics, functions, mechanisms of formation, and detection methods of ecDNA, with a focus on the potential clinical implications of ecDNA in cancer. Specifically, we consider the role of ecDNA in acquired drug resistance, as a diagnostic and prognostic biomarker, and as a therapeutic target in the context of cancer. As the pathological and clinical significance of ecDNA continues to be explored, it is anticipated that ecDNA will have broad applications in the diagnosis, prognosis, and treatment of patients with cancer.