A novel strategy for sensitive detection of biomarkers using horseradish peroxidase (HRP)-functionalized silica nanoparticles as the label is presented. The enzyme-functionalized silica nanoparticles were fabricated by coimmobilization of HRP and alpha-fetoprotein antibody (anti-AFP, the secondary antibody, Ab2), a model protein, onto the surface of SiO(2) nanoparticles using gamma-glycidoxypropyltrimethoxysilane (GPMS) as the linkage. Through "sandwiched" immunoreaction, the enzyme-functionalized silica nanoparticle labels were brought close to the surface of gold substrates, as confirmed by the scanning electron microscopy (SEM) images. Enhanced detection sensitivity was achieved where the large surface area of SiO(2) nanoparticle carriers increased the amount of HRP bound per sandwiched immunoreaction. The electrochemical and chemiluminescence measurement showed 29.5- and 61-fold increases in detection signals, respectively, in comparison with the traditional sandwich immunoassay. The improved particle synthesis using a "seed-particle growth" route yielded particles of narrow size distribution, which allowed consistent loading of HRP and anti-AFP on each microsphere and ensured subsequent immunosensing possessed high sensitivity and reproducibility. This strategy was successfully demonstrated as a simple, cost-effective, specific, and potent method to detect AFP in practical samples.