Novel green-emitting fluorescent microspheres (GreFMPs) were assembled by loading highly luminescent gold nanoclusters (Arg/ATT/AuNCs) ondendritic mesoporous silica nanoparticles (DMSNs) via PEI-mediated electrostatic adsorption. The fluorescence microspheres exhibit excellent monodispersion with average diameters about (254.5 ± 34.6nm). Compared with free Arg/ATT/AuNCs, the GreFMPs have similar fluorescent properties and biocompatibility but superior environmental tolerance. Subsequently, an immunochromatographic assay based on GreFMPs (GreFMP-ICA) has been successfully developed, which is highly selective toward alphafetoprotein (AFP) in human serum. Under the optimal parameters, there is a good linear range between 0.5 and 160.0ng/mL, the limit of detection was found to be about 0.5ng/mL, and the recovery and relative standard deviation are 81.0 ~ 100.6% and 3.5 ~ 16.6%, respectively. These results manifested that the GreFMPs are beneficial for the rational design of the ICA platform, and the proposed GreFMP-ICA has the potential to screen AFP in clinical applications.