A number of studies have shown that plasma cell-free DNA is closely related to the risk of stroke, but the fragmentation status of plasma cell-free DNA and its clinical application value in ischemic stroke are still unclear. In this study, 48 patients with new ischemic stroke and 20 healthy subjects were enrolled. The second-generation high-throughput sequencing technique was used to study the plasma cell-free fragment length and regional distribution of the subjects. As noted in our results, the ratio of plasma cell-free DNA fragments in the disease group was significantly greater than that of the healthy group in the 300–400 bp range; conversely for fragments at the 75–250 bp range, the ratio of plasma cell-free DNA fragments in the patient group was apparently lower than that of the healthy group. In-depth analysis of the proportion of fragments distributed on each component of the genome was carried out. Our results recorded that the plasma cell-free DNA fragments in the disease group were inclined to the EXON, CpG islands, and ALU regions in contrast to that of the healthy group. In particular, fragments within the 300–400 bp range of the disease group were enrichment in the regions of EXON, INTRON, INTERGENIC, LINE, Fragile, ALU, and CpG islands. In summary, our findings suggested that the intracellular DNA degradation profiles could be applied to distinguish the stroke group and the healthy group, which provided a theoretical basis for the clinical diagnosis and prognosis of stroke by profiling the characteristic of plasma cell-free DNA fragments.