Cell-free DNA (cfDNA) in human blood is currently being investigated as a minimally invasive, highly informative biomarker. Besides carrying genetic variations, cfDNA can also provide useful epigenetic information through its methylation and fragmentation pattern. Previous cfDNA studies have focused on nucleosome-sized fragments, leaving shorter ones neglected. Hence, we intended to investigate whether shorter cfDNA fragments exist in the blood or not. Using an improved cfDNA purification protocol and a 3ʹ-end labeling method, we found that human plasma contains DNA fragments of approximately 50 nucleotides, present at a molar concentration largely comparable to that of the nucleosome-sized fragments. These short fragments cannot be recovered by using popular cfDNA isolation methods, and they are composed of single-stranded DNA (ssDNA), thus escaping detection in previous studies. We established a library preparation protocol based on a ssDNA ligation technique unique developed by us and applied it to cfDNA isolated with the improved purification protocol. Deep sequencing of these libraries revealed that the short fragments are derived from hundreds of thousands of genomic sites in open chromatin regions and enriched with transcription factor-binding sites. Remarkably, antisense strands of putative G-quadruplex motifs occupy as much as one-third of these short fragments. Hence, we propose a novel class of plasma cfDNA composed of short single-stranded fragments that potentially form non-canonical DNA structures.