The proteins in the circulatory system reflect the individual's physiological and disease states. However, many protein biomarkers used for clinical diagnosis are present at very low concentration in body fluids and the detection is very difficult. In this study, we developed a fully automated protein abosolute quantification platform for simultaneous analysis of multiple tumor markers in human plasma, by which alpha-fetoprotein (AFP) , prostate-specific antigen (PSA), carcino-embryonic antigen (CEA) and mucin-1(MUC-1) were first simultaneously enriched from human plasma by aptamers immobilized capillary column with polymer@graphene oxide @polyethylenimine@Au (PM@GO@PEI@Au) modified microspheres as the matrix, followed by on-line protein denaturation, reduction, desalting and tryptic digestion by a fully automated sample treatment (FAST) device, finally the resulting peptides were analyzed by pararell reaction monitoring on a LTQ-orbitrap velos mass spectrometry. Compared to traditional ELISA assay widely used in routine clinical practice, such a platform exhibited significant advantages such as short analysis time ( 2 h vs. 12 h), low limit of detection (for AFP : 5 fg/µL vs. 46 fg/µL, and for PSA : 50 fg/µL vs. 69 fg/µL), and ease of automation. Furthermore, our developed platform was also applied in the absolute quantification of tumor markers from clinical plasma samples, and the results were comparable to those obtained by clinical immunoassay. All the results demonstrated that our developed fully automated platform should provide a promising tool for achieving high sensitivity, high accuracy, and high throughput detection of tumor protein markers in the routine physical examination and clinical disease diagnosis.