The chitosan microspheres formulated by emulsion cross-linking method were found to be smooth and spherical without aggregation. The particle size range was between 1 and 90μm. The particle sizes were found to be influenced by the concentration of the chitosan gel. Tetanus toxoide (TT) vaccine was loaded by passive adsorption from an aqueous solution into the preformed chitosan microspheres cross-linked with glutaraldehyde. The loaded TT on to microspheres was estimated by ELISA method. The loading capacity was found to be 40% with microspheres prepared with 1% chitosan gel, 43% for 2% and 46% for the mixed batch of microspheres prepared from 1% and 2% chitosan gel. The loading efficiency was found to decrease with increase in the concentration of chitosan gel. The in vitro release of the antigenic TT into the phosphate buffer at 37°C from different batch of microspheres was studied and release had a remarkable dependence on the size of micropsheres. The percentage release of TT from chitosan microspheres prepared from 1% chitosan gel was 2.7% in 120days and that from 2% chitosan gel was only 2%. The mixed batch of microspheres could release 2.3% in 120days. The antigen integrity was investigated by SDS–PAGE with brilliant blue staining. The SDS–PAGE analysis confirmed that the antigen integrity was not affected by passive adsorption of protein antigen to preformed chitosan microspheres. The study revealed that the cross-linked chitosan microspheres would be an interesting system for long-term delivery of macromolecules drugs.