Liquid biopsy of cancers using DNA methylation biomarkers has received significant interest, where the quantification of multiple biomarkers is generally needed for improving the sensitivity and specificity of cancer diagnosis. However, the inefficiency of the traditional quantitative polymerase chain reaction (qPCR)-based MethyLight assay for detecting the extremely low concentration of methylated DNA fragments in body fluids limits its clinical applications. Here, we developed an ultrasensitive microwell chip digital polymerase chain reaction (dPCR)-based MethyLight assay. Using the synthesized samples, the developed MethyLight assay can achieve 103-104-fold lower limit of detection and 1-16-fold lower limit of quantification than the traditional MethyLight assay. Four hypermethylated alleles (RARβ2, BRCA1, GSTP1 and RASSF1A) related to breast cancer in twenty-three clinical samples were tested using the microwell chip dPCR-based MethyLight assay. The results showed that the dPCR assay achieves ∼2 times enhancement in the cancer detection rate over the traditional quantitative PCR. Furthermore, the dPCR can detect the healthy and benign samples, which are undetectable using the traditional MethyLight assay. In multiple gene analysis, we achieved the highest detection rate of 93.3% (in the "OR" format of RARβ2 and GSTP1). Lastly, the estimated cut-off values in the dPCR assay were: <1, ∼1 to 100 and >100 (copies per μL) referring to the healthy, benign and malignant breast cancers, respectively. Therefore, the developed microwell chip dPCR-based MethyLight assay could provide a powerful tool for cancer biopsy diagnosis and disease monitoring.