The analysis of cell-free DNA (cfDNA) in plasma represents a rapidly advancing field in medicine, providing information on pathological processes in the body. Blood cfDNA is in the form of nucleosomes, which maintain their tissue- and cancer-specific epigenetic state. We developed a single-molecule multi-parametric assay to comprehensively profile the Epigenetics of Plasma Isolated Nucleosomes, DNA methylation and cancer specific protein biomarkers. This analysis is combined with direct single-molecule DNA sequencing, providing multi-layered clinical-relevant information from limited liquid biopsy material, establishing a novel approach for cancer diagnostics. We aim to expand the collection of analytes used by our method, as well as generalize it for analysis of multiple types of tumors.