A simple and high-efficiency approach to loading macromolecules into microscale carriers is presented. Calcium-cross-linked alginate hydrogel microspheres were fabricated by an emulsification technique and then used as negatively charged templates to form polyelectrolyte multilayer coatings. A calcium ion chelator, EDTA, was used to free the Ca2+-cross-linked alginate hydrogel within {poly(allylat-nine hydrochloride)/ poly (styrene sulfonate)}(4) ({PAH/PSS}(4)) coating, allowing partial release of alginate. The retention of alginate in {PAH/PSS}(4) inicrocapsule was confirmed by FTIR spectroscopy and confocal microscopy. Real-time confocal microscopy was used to investigate the loading process of positively charged macromolecules (dextran-amino, and peroxidase) into alginate-templated inicrocapsules, which showed the loading occurred in < 2 min for dextran-amino and < 10 min for peroxidase, respectively. A high loading efficiency of 25 mu g peroxidase in similar to 1.0 x 10(7) microcapsules (2.5 pg POx/capsule) was achieved with a low concentration of peroxidase loading solution (10 mu g/mL). This spontaneous loading technique for encapsulating positively charged molecules in alginate-templated polyelectrolyte microcapsules shows strong potential for biosensor and drug delivery applications.