Three-dimensional microspheres have been used extensively in several biomaterials fields, in applications such as tissue-regeneration scaffolds and drug delivery systems. To apply these biomaterials as novel cell therapeutic agents, we have devised a novel method for the fabrication of nanostructured 3D scaffolds consisting of heparin/poly(l-lysine) nanoparticles on the surface of polymeric microspheres, attached via a layer-by-layer (LbL) system. The initial step of this strategy involves the creation of heparin/poly(l-lysine) nanoparticles, which were simply produced as polyion complex micelles (PICM) with diameters of 200-500 nm. In the second step, the heparin/poly(l-lysine) nanoparticles were coated onto positively charged poly(lactic-co-glycolic acid) (PLGA) pretreated with polyethyleneimine (PEI). The production of the heparin/poly(l-lysine) nanoparticles and their subsequent coating onto PLGA microspheres represents a novel method for the functionalization of the polymeric matrix, which requires the cellular active surfaces of nanoscaled heparinized surfaces as 3D scaffolds, thus creating a better microenvironment of cell adhesion and growth for use in cell therapy applications.