Poly(l-lactide-co-glycolide) (PLGA) microspheres with diverse internal structures and different release behaviors were prepared via a modified double-emulsion method by introduction of heparin or carboxymethyl chitosan in the inner aqueous phase and calcium chloride in the outer aqueous phase, respectively. The main factors affecting the microsphere morphology were systematically studied, including compositions in the inner aqueous phase, the oily phase, and the outer aqueous phase. The transmission electron microscope images demonstrated that the microspheres are featured with single core, hollow, and multicore structures when their sizes were less than 200 nm, in the range of 200-700 nm, and greater than 700 nm, respectively. In comparison with hollow PLGA microspheres, the PLGA microspheres with heparin and carboxymethyl chitosan in the inner aqueous phase also showed multicore and single core structures, respectively, and exhibited higher loading efficiencies and slower release rates by using bovine serum albumin as a model for bioactive substances. It was concluded that this study provided a facile method to prepare microspheres with single core, multicore, or hollow feature, and the tunability of the different internal structures and related release profiles enables these systems cater to specific requirements for potential applications in controlled biomolecule delivery for tissue regeneration. POLYM. ENG. SCI., 55:896-906, 2015. (c) 2014 Society of Plastics Engineers