Defective viral genomes (DVGs) are truncated or rearranged products derived from parental viruses during replication and they harbour numerous lethal mutations or large-fragment deletions. Current studies have demonstrated that DVGs play crucial roles in interfering with normal viral replication and activating host innate immunity, suggesting that the presence of DVGs not only may modulate the course of viral infection but also holds substantial potential for applications in antiviral drugs and immune adjuvants. Nevertheless, the mechanisms underlying the functions of different types of DVGs remain incompletely elucidated, thus emphasising the necessity of updating analytical and research methodologies. Consequently, while summarizing the core functions, mechanisms of action, and the evolution of DVGs, this review provides a detailed overview of their classification methods to shed light on the characteristics of DVG formation and functions. In addition, it reviews the current detection technologies of DVGs to enable the identification and accurate quantification of DVGs. Furthermore, the discussion encompasses the distinctive mechanisms of action and the potential applications of DVGs derived from diverse viruses, with the objective of providing novel insights into the development of clinical antiviral drugs.