Highly sensitive and specific detection of DNA methylation is critical for early diagnosis and therapy of cancer. Herein, we propose a novel bisulfite-free PCR assay based on a GlaI methylation specific digestion and terminal transferase (TdT) extension for the detection of methylated DNA with high sensitivity and specificity, denoted as GlaI-TdT methylation PCR. For GlaI-TdT methylation PCR assay, the methylated CpG site is recognized and cut by GlaI selectively firstly, leading to the generation of product with specific free 3' end. The free 3' end can be further extended with TdT and served as template for the followed quantitative PCR. The specificity of GlaI-TdT methylation PCR depends on the specific methylation discrimination of GlaI and the existence of poly-T sequence as the extension of TdT. The sensitivity of GlaI-TdT PCR for methylated DNA can achieve 10 copies/reaction with 10,000 copies unmethylated background. The detection performance of GlaI-TdT methylation PCR was also evaluated using colorectal cancer tissue samples, with the results shown great accordance with standard bisulfite-PCR sequencing. Based on its high sensitivity, high specificity, simple and convenient, GlaI-TdT methylation PCR has the great potential to become a promising and robust bisulfite-free procedure for the detection of DNA methylations.