Glatiramer acetate (GA, Copaxone, COP, Copolymer-1) is one of the most prescribed drugs to treat relapsing-remitting multiple sclerosis (RRMS). Herein, the formula of GA entrapped chitosan-alginate-poly (D, L-lactic-co-glycolic acid) (CS-Alg-PLGA) microsphere has been studied as a novel carrier compared with conventional PLGA microsphere, pursuing long-lasting release to reduce the number of injections and minimize side effects. The preparation methods, including double-emulsion, homogenization, and solvent evaporation, were evaluated in preparing the GA/PLGA and GA/CS-Alg-PLGA microspheres. Dynamic light scattering (DLS), Field Emission Scanning Electron Microscopes (FE-SEM), Fourier transform infrared spectroscopy (FTIR), 1H nuclear magnetic resonance (1H NMR), and Thermogravimetric analysis (TGA) were performed for microspheres characterization. GA was successfully loaded within the PLGA and CS-Alg-PLGA microspheres with 96.93 ± 0.9% and 97 ± 0.86% encapsulation efficiency, with 96.2 ± 1.24% and 86.91 ± 1.18% in vitro release in 21 days, respectively. Moreover, the cell toxicity assay showed that the PLGA and CS-Alg-PLGA microspheres with and without the GA drug were nontoxic (at the range of this experiment) for Human Dermal Fibroblasts (HDF) cell line. In vitro release experiments exhibited long-lasting release of GA from both microspheres. Using the GA/PLGA and GA/CS-Alg-PLGA microspheres in treating RRMS may improve the therapeutic efficiency and bioavailability of the drug.