A novel fluoroimmunoassay (FIA) method was developed for the determination of tumor necrosis factor-alpha (TNF-alpha) in this study. The proposed method has the advantage of showing the specificity of immunoassays and sensitivity of fluorescent nanoparticles label technology. With the well-established inverse microemulsion polymerisation process, the tris(2',2-bipyridyl)dichlororuthenium(II) hexahydrate (Rubpy)-doped fluorescent silica nanoparticles (RuDFSNs) were prepared. Then a RuDFSNs-labeled anti-TNF-alpha monoclonal antibody was prepared and used for FIA of TNF-alpha in human serum samples with a sandwich FIA by using the low fluorescent 96-well transparent microtiter plates. The assay response was linear from 1.0 to about 250.0pg/mL with a detection limit of 0.1pg/mL for TNF-alpha. The intra- and inter-assay precision are 4.9%, 4.4%, 4.6%; 6.1%, 5.9%, 5.3% for five parallel measurements of 2.0, 20.0, 200.0pg/mL TNF-alpha respectively, and the recoveries are in the range of 96-104% for human serum sample measurements by standard-addition method. We also explored the application of fluorescence microscopy imaging in the study of the FIA for TNF-alpha with the fluorescent nanoparticle labels. The results demonstrate that the method offers potential advantages of sensitivity, simplicity and good reproducibility for the determination of TNF-alpha, and is applicable to the determination of TNF-alpha in serum samples and being capable of fluorescence microscopy imaging for the determination of TNF-alpha.