Fluorescent polystyrene microparticles with different sizes were produced by the consecutive assembly of fluorescently labeled polyelectrolytes using the layer-by-layer self-assembly method. Film growth was characterized by microelectrophoresis and fluorescence microscopic image (FMI) analysis. Alternating negative and positive ζ-potentials with deposition of each successive polyelectrolyte layer demonstrated that the alternate adsorption of polyelectrolytes was achieved. FMI analysis provided direct measurement of the fluorescence intensity of single microparticles. The subsequent deposition of a protein (immunoglobulin G, IgG) layer onto the fluorescent microparticles was confirmed by a sandwich immunoassay.