Analysis of cell-free DNA (cfDNA) in the blood has shown promise for monitoring a variety of biological processes. Plasma cfDNA is a mixture comprising DNA molecules released from various bodily tissues, mediated by characteristic DNA fragmentations occurring during cell death. Fragmentation of cfDNA is non-random and contains tissue-of-origin information, which has been demonstrated in circulating fetal, tumoral, and transplanted organ-derived cfDNA molecules. Many studies have elucidated a plurality of fragmentomic markers for noninvasive prenatal, cancer, and organ transplantation assessment, such as fragment sizes, fragment ends, end motifs, and nucleosome footprints. Recently, researchers have further revealed the large population of previously unidentified long cfDNA molecules (kilobases in size) in the plasma DNA pool. This review focuses on the emerging biological properties of cfDNA, together with a discussion on its potential clinical implications.