A direct and ultrasensitive multiplex assay using an immuno-magnetic platform has been developed for the quantification of trace amounts of circulating cancer-associated antigens in serum. The detection is based on the specific immuno-interactions among the target antigen, detection antibody and capture antibody that is immobilized on the surface of magnetic nanoparticles. The sandwiched immuno-assembly is then labelled with turn-on fluorophores and detected with a fluorescence imaging system. To afford a high signal-to-noise ratio, three turn-on fluorophores with unique optical properties have been designed and synthesized to label the target antigens. The developed assay has achieved a remarkable LOD down to the femto-molar regime without sample pre-treatment. This versatile assay can efficiently differentiate the target antigen from a protein matrix and simultaneously quantify multiple cancer-associated antigens, for instance, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and prostate specific antigen (PSA) using only 6 μL of serum sample in an hour. This novel system has a high applicability to serve as a universal and useful tool for early disease diagnostics.