Polysaccharide multilayer microcapsules were fabricated in aqueous media by the layer-by-layer self-assembly of chitosan (CHI) and sodium alginate (ALG) on melamine formaldehyde (MF) microparticles of 2.1μm diameter as templates, followed by removal of the templates through dissolving at low pH. The loading process was observed with the confocal laser scattering microscope (CLSM) using fluorescence labeled insulin. Insulin was spontaneously loaded into the ALG/CHI microcapsules at pH below its isoelectric point of 5.5 where insulin was positively charged and the loading capacity increased with pH decreasing from 4.0 to 1.0. Because there exited a negatively charged complex of ALG/MF residues inside the microcapsules formed during the MF particle dissolution. A novel two-temperature loading procedure was proposed as loading at 20°C for the first hour and at a higher loading temperature for the second hour. This procedure was very significant that increasing the second loading temperature from 20 to 60°C not only increased the insulin loading capacity, but also slowed down its release rate. The release rate of insulin at pH 7.4 was found much faster than that at pH 1.4 due to the positive charges on the insulin. Cross-linking the ALG in the microcapsule shell with calcium ions (Ca2+) or re-sealing the microcapsules with additional layers also remarkably decreased the insulin release rate. The results provide a simple method to control the loading and release of protein molecules within these polysaccharide microcapsules.