This study devised and examined a sandwich-like assay for detecting vitamin D3 dissolved in acetonitrile using fluorescent dye-embedded silica nanoparticles (AF647@SiNPs). First, AF647@SiNPs were synthesized via the modified Stöber method to embed the fluorescent dye molecules in the silicate network. Then, vitamin D3 (sample) was immobilized using an azide linker on the magnetic nanoparticles (MNPs). The immobilized vitamin D3 reacted with the antibody immobilized on the surface of AF647@SiNPs (Anti-VD3@AF647@SiNPs). The vitamin D3 concentration was calculated back using the fluorescence of the remaining unreacted Anti-VD3@AF647@SiNPs. Finally, we examined how applying conditions of AF647@SiNPs influenced the measurement reliability: (1) the initial loading of AF647@SiNPs; (2) the antibody-labeling degree of AF647@SiNPs.