Fluorescent microspheres are greatly demanded in many applications based on high-throughput suspension array technology. To realize the multiplexed assay, microspheres should be encoded to identify the interaction between analytes and spheres. This study advanced a strategy for preparing fluorescence-encoded microspheres, employing two hydrophobic fluorophores, poly(p-phenyleneethylene) (PPE), and Nile Red (NR), as well as the monodisperse amino-modified porous substrate polymeric spheres, poly(glycidyl methacrylate) microspheres (APGMA). Loading the fluorophores sequentially onto the substrate spheres via adsorption by immersing the spheres in the dipping solution of fluorophores resulted in the APGMA-PPE-NR spheres. By varying the concentration and combination of fluorophores in the solution, an array of 64-code APGMA-PPE-NR spheres was obtained and could be easily individually decoded via flow cytometry. A 2D dot plot from the flow cytometry of a set of mixed spheres with four different codes could also be differentiated, coincident with the overlaid plots of the spheres' corresponding codes but measured individually. These spheres were found to have good stability against washing, photobleaching, and thermal treatment. In addition, a sandwich immunoassay for the detection of goat IgG was performed, and the capability of the encoded spheres to be used in suspension array technology was demonstrated.