Chromatin accessibility refers to the level of physical compaction of chromatin, which is determined by the chromatin binding factors that hinder DNA contact, nucleosome occupancy and topological structure. The chromatin accessibility pattern will be changed dynamically with external stimuli and developmental cues. Analyzing the TF (transcription factor) binding sites in the regulatory regions within the accessible chromatin can provide insight into the lineage factors and gene regulatory networks of specific cell types. Combined with high-throughput sequencing technology, several biochemical methods have been developed to describe the accessibility of chromatin, including bulk and single-cell level analysis. Depending on the techniques, the using enzymatic cleavage (DNase/MNase), transposition (Tn5) or physical methods (FAIRE) to isolate the accessible chromatin and subsequently using the high-throughput sequencing provide a genome-wide panorama of chromatin organization. This review introduced the common techniques (DNase-seq, MNase-seq, FAIRE-seq, and ATAC-seq) for determining chromatin accessibility and nucleosome positioning. The advantages and disadvantages of these 4 chromatin accessibility analysis techniques were summarized and compared. Their principles and main experimental procedures were introduced in detail; the development and application of related technologies were briefly discussed. The ATAC-seq based single-cell chromatin accessibility analysis and the view of potential useful were specially introduced. Although the chromatin accessibility profile is very valuable for studying gene regulation, it only provides a partial view of this complex process. We envision that technological improvements including single-molecule, multi-omics and spatial methods will bring further insight into the secrets of genome regulation.