Extracellular vesicles (EVs) have recently emerged as pivotal mediators of intercellular communication, influencing disease progression in liver failure. EVs can carry diverse molecules, including proteins, RNAs, and linear double-stranded DNAs. However, the presence of extrachromosomal circular eccDNA (eccDNA) within EVs, as well as its potential relationship with the development of liver failure, remains to be definitively understood. In this study, we isolated and investigated eccDNA from plasma EVs of both liver failure patients (LFEVs-eccDNA) and healthy control individuals (HCEVs-eccDNA), comparing their characteristics between the two groups and conducting eccDNA functional assays. The findings demonstrated that LFEVs-eccDNA exhibited increased abundance, shorter length, and carried more coding genes, transposon genes, and cis-regulatory elements. Furthermore, LFEVs carried eccDNA (LFEVs-eccDNA) containing more segments of liver-specific expression genes. Additionally, through comparative analysis of eccDNAs with identical start-end sites between the two groups, we identified the over-represented eccDNAs in LFEVs, including eccZMIZ1-AS1 and eccZMYM6. Functional analysis through artificial eccDNA transfection and RNA-seq in HepG2 cells revealed that the introduction of synthetic eccZMIZ1-AS1 significantly activated the PI3K-Akt and HIF-1 signaling pathways, while eccZMYM6 had no influence on the RNA profile. Moreover, eccZMIZ1-AS1 prominently promoted the cell response to hypoxia, regulated lipid metabolism, and was related to vesicle formation. Together, our study revealed aberrant eccDNA hallmarks in plasma EVs of liver failure patients and suggested that over-represented LFEVs-eccDNA may exacerbate liver damage by disrupting the transcriptome of hepatocytes. It provides a potential noninvasive biomarker for diagnosing and monitoring liver failure. Moreover, targeting the LFEVs-eccDNA may present promising therapeutic strategies for treating liver failure.