Extrachromosomal circular DNA (eccDNA) is a class of circular DNA molecules that originate from and are independent of conventional chromosomes. The high specificity and stability of the circular structure of eccDNA provide new clues for its research as a disease biomarker. The recent rapid development of long-read sequencing and eccDNA identification algorithms has greatly expanded our understanding of eccDNA properties. In this review, we introduce the molecular characteristics, biological origin, and advances in detection technology of eccDNA, and mainly summarize the clinical implications of eccDNA in cancer diagnosis and prognosis, drug resistance monitoring, prenatal testing, and immune-related disease, providing support for further exploring new clinical applications of eccDNA.