Provided are a fluorescent microsphere comprised of a plurality of fluorescent nanocrystals embedded in a polymeric microsphere; a kit prepared from the fluorescent microspheres; and a method of producing the fluorescent microspheres comprising swelling the polymeric microsphere so that into its pores can enter fluorescent nanocrystals, and then unswelling the polymeric micropsheres so that the fluorescent nanocrystals become physically entrapped in the pores of the unswelled polymeric microsphere. Also provided is a method of using the fluorescent microspheres comprising affinity ligand for determining the presence or absence of a predetermined number of analytes in a sample by contacting the sample with the fluorescent microspheres, and detecting the fluorescence signal pattern of excited fluorescent microspheres bound to one or more analytes of the predetermined number of analytes, if present in the sample.