A novel method with two steps for preparing high efficiency fluorescence nanocrystals (NCs)-encoded microspheres was developed. First, a series of polystyrene (PS) microspheres from 1 μm to 5 μm in diameter were prepared by dispersion polymerization; then, after a swelling process with CdSe NCs of different size the PS microspheres were encoded quantitatively. This method for fluorescence-encoding of microspheres was shown to have the following outstanding characteristics: (1) An emulsion containing CdSe NCs and swelling agents (chloroform) are firstly used for introduction of NCs into microspheres. (2) The embedded nanocrystals remain their original properties and they are not simply absorbed onto the surface of the microspheres but being carried into the inner of microspheres and so the NCs cannot be released or leaked out easily. As-prepared fluorescence-encoded microspheres could conjugate with proteins by some further surface modification, so they have a great potential to be applied to such fields as biochemistry, molecular biology, cell biology, immunology, etc. In addition, the method could be operated easily and reproducibly.