Polyelectrolyte multilayer microcapsules have been prepared for encapsulating horseradish peroxidase (HRP). The CaCO3 microparticles containing HRP molecules were prepared and subsequently enwrapped with layer-by-layer (LbL) deposited multilayer films. The LbL films were constructed via an alternate electrostatic adsorption of poly(allylamine) and poly(styrene sulfonate). The CaCO3 templates were then dissolved in an ethylenediaminetetraacetic acid solution, leaving HRP in the microcapsules. Optical microscope and fluorescence microscope observations indicated that the microcapsules are homogeneous spheres with an average diameter about 7 μm. The encapsulated HRP showed catalytic activity in the oxidation of pyrogallol, suggesting the microcapsules provide a suitable environment for enzymatic reaction. A leakage of HRP from the microcapsules was negligibly small. Therefore, the HRP-containing microcapsules is promising for detection and treatment of phenolic compounds.