DNA methylation discrimination is often challenged by complicated pretreatment, insufficient sensitivity, and suboptimal accuracy. Here, single-molecule readout of DNA methylation is reported using single-layer MoS2 nanopores. By tuning pore dimension, the sensitivity of MoS2 nanopores is manipulated, empowering both labeling and labeling-free strategies for DNA methylation discrimination. With methyl-CpG-binding domain protein 1 (MBD1)-labeled methylated DNA translocation in customized nanopores, multiple methylated sites with distance as short as 70 bp in double strand DNA can be resolved. To further improve spatial resolution, small MoS2 nanopores are engineered with single-nucleotide sensitivity, realizing labeling-free methylation detection with single-nucleotide resolution to recognize two nucleotides with only one methyl difference. This study demonstrates the availability of engineered MoS2 nanopores in DNA methylation detection, underscoring their potential for epigenetic alteration research at the single-molecule level.