IgG responses to antigen-nanosphere hybrids were studied in mice. Cholera toxin (CT) was covalently immobilized onto the surface of polymeric nanospheres (NS) with a nanophase-separated structure consisting of a polystyrene core and a poly(methacrylic acid) graft corona. Reaction conditions favoring the dehydroxide condensation reaction of the amino group of the CT with the carboxyl group of NS effectively immobilized CT onto their surface. When CT-immobilized nanospheres (CT-NS) were suspended in aqueous solution and administrated to mice either intranasally or intramuscularly, serum IgG titers elevated with increasing time and reached a maximum level at 8 weeks after immunization. On the other hand, intranasal administration of CT alone induced an even higher serum IgG titer than that of CT-NS at 4 weeks. However, the titer gradually decreased thereafter. Thus, polymeric NS may be an effective substrate to covalently immobilize antigen on their surface, steadily inducing a high level of IgG production in response to the intranasal administration.