Here we present a single-cell 3D genome mapping assay with improved spatial resolution with the help of micrococcal nuclease (MNase), termed single-cell Micro-C (scMicro-C). To achieve scMicro-C, we made three improvements. First, we titrated MNase digestion to reduce DNA loss and produce proper DNA fragments. Second, we solubilized chromatin with an ionic detergent, sodium dodecyl sulfate (SDS), which dramatically improved ligation efficiency. Third, we adopted our high-coverage transposon-based whole-genome amplification method, META (19), using Tn5 (20), to enhance the detection of chromatin “contacts.” scMicro-C retains the high signal-to-noise and high-resolution characteristics of bulk Micro-C, as indicated the accurate detection chromatin loops and other fine-scale chromatin structures. Furthermore, scMicro-C retains nucleosome and other chromatin-bound proteins (e.g., transcription factor) occupancy information. With our previously developed Dip-C algorithm, we confirmed that scMicro-C enables the reproducible reconstruction of 3D genome structures in single-cell at 5 kb resolution. With such kilobase 3D structures, we were able to explore the fine-scale chromatin folding in individual cells.