Controlled synthesis of spinel ZnFe2O4 nanoparticle-decorated ZnO nanofiber heterostructures was carried out with regularly varied particle sizes and uniform distribution. The obtained heterostructures possessed enhanced intrinsic peroxidase-like activity, and the activities regularly changed as the sizes of ZnFe2O4 nanoparticles varied. The heterostructures were further applied in sensitive colorimetric biosensing of urine glucose.