Nanometer-sized europium-doped calcium sulfide (CaS–Eu) particles were synthesized for the first time for potential use in bioassays, in particular, in biochip-based analysis. The CaS–Eu nanoparticles were prepared through a wet chemical process in ethanol. The average diameter of the nanoparticles was about 15 nm. They were characterized by UV–Vis spectrometry, scanning electron microscopy, X-ray diffraction, fluorescence spectroscopy and X-ray sequential fluorescence spectroscopy. Both electron paramagnetic resonance measurement and an extensive washing experiment revealed that the dopant atoms were internal ions dispersed throughout the whole CaS–Eu nanoparticles. Factors affecting the fluorescent properties of the nanoparticles were examined. It was found that the emission wavelength of the CaS–Eu nanoparticles could be altered by partial replacement of calcium with other alkaline earth metals such as magnesium, strontium and barium, and this makes the nanoparticles ideal for use as biochips where a multicolor-based bioassay is common.