DNA methylation (5-methylcytosine, 5mC) represents the most prevalent modification in mammals, which is closely linked to disease pathogenesis and cancer development. Single-base resolution sequencing and quantitative analysis of 5mC are essential for elucidating its biological functions. However, current methods are still limited by resolution, sequencing bias, and false positives. In this study, we engineered a Naegleria TET-like protein (NTET) , yielding a recombinant engineered NTET (eNTET), to improve both its oxidation activity and sequence compatibility for 5mC. Combined with pyridine borane reduction, we developed engineered NTET-assisted pyridine borane sequencing (eNAPS) to quantitatively detect 5mC in DNA at single-base resolution. In eNAPS, 5mC is oxidized by eNTET to 5‑formylcytosine (5fC) and 5-carboxylcytosine (5caC), which are further reduced to dihydrouracil (DHU) by pyridine borane and read as thymine (T) in the subsequent sequencing. The direct conversion of 5mC-to-T allows for precise mapping of 5mC in DNA at single-base resolution. Compared with conventional bisulfite sequencing (BS-seq), eNAPS exhibits advantages such as non-destruction, enhanced sensitivity, improved accuracy, and greater efficiency. Using the eNAPS method, we achieved quantitative analysis of 5mC at single-base resolution in genomic DNA of lung tumor and tumor-adjacent normal tissues. Overall, eNAPS is a mild and bisulfite-free method with high accuracy, making it a valuable tool for investigating the dynamic interplay of 5mC in epigenetic regulation and disease pathogenesis.