This study advanced a strategy for preparing fluorescent spectrally encoded microspheres through introducing rare earth element europium (Eu) and organic dye rhodamine 6G (R6G) onto the porous substrate polymer spheres. The size and monodispersity of the microspheres were controlled by selecting the appropriate substrate spheres. Various combinations of three different types of fluorophores, the substrate spheres as a polymer fluorophore, together with Eu and R6G as inorganic and organic fluorophores afford large encoding capacity based on emission spectra. After optimization of the preparative process, three series of fluorescent spheres with ten combinations of intensities at different wavelengths from two binary encoding systems and fifteen combinations from one ternary encoding system were prepared. Such combinations (or codes) were obtained by exciting the spheres at the same wavelength. The preparative process was found to have no significant influence on the size and surface morphology, as shown in the microscopic study. The color variation in the macroscopic and microscopic images under excitation was consistent with emission spectra. In addition, these spheres were found to have good stability against water washing and thermal treatment.