The biogenesis and functions of extrachromosomal circular DNA (eccDNA) have been studied for several decades. However, the heterogeneity of eccDNA is largely ignored. In this study, we purified and sequenced eccDNA and RNA using a method that simultaneously extracts DNA and RNA from cultured cells treated with iron nanoparticles. We discovered that the characteristics of eccDNA are altered by iron stress, particularly on genic and CpG islands regions, suggesting a biogenesis mechanism induced by transcription. However, we found that only a few eccDNA-annotated genes were shared by differentially expressed genes identified by RNA-seq, indicating that eccDNA has a complex background that may be the result accumulated from previous states. In addition, we observed little variation in the distribution pattern of eccDNA in ferroptosis cells with fewer differentially expressed genes, indicating that eccDNA is generated randomly in cells when they are unable to regulate many more genes to resist environmental stress. Collectively, our study characterized eccDNA induced by iron stress, uncovered their intricate background, and proposed the alterations in the distribution pattern of eccDNA as a potential biomarker.