Our laboratory develops a method of microencapsulation using a transacylation reaction in a water-in-oil (W/O) emulsion. The method is based on the creation of amide bonds between free amine functions of a protein (human serum albumin (HSA)) and ester groups of propylene glycol alginate (PGA) in the inner aqueous phase after alkalization. The aim of this work is to study the influence of physicochemical properties of HSA–PGA mixtures on microparticle characteristics. Microparticles were prepared varying the concentrations of PGA and HSA, then characterized (inner structure, size, swelling rate, release kinetics). PGA and each polymer mixture used in the microencapsulation procedure were examined in order to elucidate the mechanism of microstructure formation. It was found that the morphology and functional properties of HSA–alginate microparticles were related to the two polymer concentrations in the aqueous solution. Actually, the polymer concentration variations led to physicochemical changes, which affected the microparticle structure and functional properties.