Abstract: AIM To evaluate the immunogenicity of tetanus toxoid (TT) encapsulated in biodegradable polymer microspheres composed of polylactide (PLA). METHODS The antibody levels elicited by microsphere formulations in mice for 1 year were examined, the anamnestic responses to a low dose booster 1 year after priming were also examined. RESULTS Microsphere formulations made from PLA were characterized by pulse controlled-release models, differing in polymer molecular weight, protein loading and particle size of the microspheres. Microsphere formulations elicited significantly higher IgG antibody levels than a single injection of soluble TT. The antibody levels elicited by microsphere formulations were similar to those elicited by three doses of soluble TT. CONCLUSION A single-dose tetanus toxoid based on pulsed release from biodegradable and biocompatible polymer microspheres has been developed. The formulations showed great benefits and pharmaceutical application.