Nucleosome organization is important for chromatin compaction and accessibility. Profiling nucleosome positioning genome-wide in single cells provides critical information to understand the cell-to-cell heterogeneity of chromatin states within a cell population. This protocol describes single-cell micrococcal nuclease sequencing (scMNase-seq), a method for detecting genome-wide nucleosome positioning and chromatin accessibility simultaneously from a small number of cells or single cells. To generate scMNase-seq libraries, single cells are isolated by FACS sorting, lysed and digested by MNase. DNA is purified, end-repaired and ligated to Y-shaped adaptors. Following PCR amplification with indexing primers, the subnucleosome-sized (fragments with a length of ≤80 bp) and mononucleosome-sized (fragments with a length between 140 and 180 bp) DNA fragments are recovered and sequenced on Illumina HiSeq platforms. On average, 0.5-1 million unique mapped reads are obtained for each single cell. The mononucleosome-sized DNA fragments precisely define genome-wide nucleosome positions in single cells, while the subnucleosome-sized DNA fragments provide information on chromatin accessibility. Library preparation of scMNase-seq takes only 2 d, requires only standard molecular biology techniques and does not require sophisticated laboratory equipment. Processing of high-throughput sequencing data requires basic bioinformatics skills and uses publicly available bioinformatics software.