5-Methylcytosine (5mC) is the most significant DNA modification present in mammalian genomes. Understanding the roles of 5mC in diverse biological processes requires quantitative detection at single-base resolution. In this study, we engineered the repressor of the silencing 1 (ROS1) protein derived from Arabidopsis thaliana to enhance its 5mC glycosylase/lyase activity, resulting in the creation of the engineered ROS1 (eROS1) protein. Leveraging the unique properties of eROS1, we introduced a method termed engineered ROS1 sequencing (eROS1-seq) for bisulfite-free and quantitative detection of 5mC in DNA at single-base resolution. In eROS1-seq, the eROS1 protein selectively cleaves 5mC while leaving unmodified cytosine (C) intact, followed by the incorporation of dTTP, which subsequently results in sequencing as thymine (T). This method effectively differentiates between C and 5mC. Unlike conventional bisulfite sequencing (BS-seq), which predominantly converts cytosines, eROS1-seq specifically transforms 5mC into T, thereby avoiding potential imbalances in the nucleobase composition of the sequencing library. Using eROS1-seq, we successfully achieved quantitative and site-specific detection of 5mC in the genomic DNA of lung cancer tissue. Overall, the eROS1-seq approach is bisulfite-free and straightforward, making it a valuable tool for the quantitative detection of 5mC at single-base resolution.