DNA is continuously exposed to both endogenous and exogenous damaging agents, resulting in the production of various DNA damages. DNA damage could compromise genome integrity and adversely affect various cellular processes, including transcription, replication, and chromatin assembly. Accurate mapping of DNA damage is crucial for elucidating its role in both biological and pathological contexts. In this study, we propose a dual enzyme cleavage-assisted sequencing (DEC-seq) method for genome-wide mapping of DNA damage at single-nucleotide resolution. DEC-seq integrates damage-specific glycosylase excision with restriction endonuclease cleavage to selectively enrich DNA containing damage while effectively eliminating background DNA without damage. We successfully applied DEC-seq to map uracil (U), 8-oxo-7,8-dihydroguanine (8OG), and apurinic/apyrimidinic (AP) sites in DNA at single-nucleotide resolution. The genome-wide mapping analysis revealed that uracil is enriched in centromeric DNA. Notably, DEC-seq is an antibody-free and chemical labeling-free method, which facilitates precise location analysis of various types of DNA damage using the corresponding glycosylases. Moreover, DEC-seq addresses the limitation of requiring specific methods for each type of DNA damage by providing a universal sequencing platform suitable for analyzing a broad spectrum of DNA damages and rare DNA modifications. Collectively, DEC-seq offers a high-resolution, broadly applicable, efficient, and label-free approach for mapping DNA damages and modifications within genomes.