The invention relates to interdisciplinary fields of biology, medicine, material, chemistry and virology, and discloses a homogeneous immunoassay based method for synchronous fluorescence detection of multiple disease markers. The method comprises the specific steps of: first, covalently coupling different fluorescent dyes with antibodies of different disease markers, and dissolving in water or other solvents to obtain a reaction solution A; second, mixing the solution A with a graphene oxide solution to obtain a reaction solution B; third, mixing the reaction solution A, the reaction solution B and water or other solvents to obtain a solution C; and fourth, adding an antigen solution into the mixed solution C, transferring the mixed solution to a fluorescence cuvette, measuring synchronous fluorescence intensity, and conducting quantitative analysis and multi-index analysis and diagnosis. The method has advantages of simpleness, rapidness, high sensitivity, high specificity and low cost, and can realize high-throughput detection, multiple index analysis diagnosis and remote diagnosis.