Abstract: It is disclosed a method for simultaneous detection of multiple tumor markers, comprising the steps of: (a) mixing analyte samples, a first antibody solution and a second antibody solution, thereby forming a quaternary composite of “Second Antibody-Tumor Marker-First Antibody-bead”; (b) detecting the detectable signals from the distinct beads of the quaternary composites to determine the presence of each tumor marker in the analyte sample. The method of invention can simultaneously detect multiple tumor markers qualitatively or quantitatively. It is also simple, rapid and accurate. The invention also provides the corresponding assay kit.