Leptospirosis is one of the most prevalent zoonotic diseases worldwide. Currently, multivalent whole-cell leptospiral vaccines can induce protection against leptospirosis. Therefore, preparation and formulation of new generations of vaccines that can stimulate long-term immunity for leptospirosis control are essential. The aim of this study was to prepare and characterize alginate microspheres as an antigen delivery system for immunization against leptospirosis. We used five Leptospira interrogans serovars, namely, Icterohaemorrhagiae, Grippotyphosa, Serjo harjo, Pomona, and Canicola, for antigen preparation. Alginate microspheres containing leptospiral antigen (LA) were prepared by an emulsification method and evaluated with respect to morphology, size distribution, loading efficiency (LE), loading capacity (LC), and release profile. The effects of concentration of alginate and emulsifiers and stirring rate on characteristics of microspheres were investigated. The optimal condition parameters for the preparation of LA-loaded alginate microspheres were estimated. The optimum concentrations obtained for alginate and emulsifiers were 3.65% (w/v), Span 80 (0.24% w/v), and Tween 80 (3.85% w/v), respectively. Moreover, the appropriate homogenization rate was 500 rpm. Our results showed mean particle size of 200 μm, 97.41% LE, and 8% LC for the microspheres. Sufficient release profile was observed for in-vitro release test of LA from alginate microspheres over an extended period of time (216 hour). Therefore, alginate microspheres technologically seem to be a promising antigen delivery system for leptospiral vaccine.