A well-defined poly[styrene (S)-b-2-vinylpyridine (2VP)] diblock copolymer was prepared by sequential anionic addition. The polystyrene (PS) block part of this diblock copolymer contained a small amount of isoprene (I) sequence units at the prescribed position. The crosslinking reaction of the block copolymer film with poly(2-vinylpyridine) (P2VP) spherical microdomains was carried out with 1,4-diiodobutane (DIB) vapour. Electron micrographs and particle size distribution of this crosslinking product showed the microstructure of core-shell type polymer microspheres. Cores and shells were formed by crosslinked P2VP blocks and PS branchings, respectively. Subsequently, ozonolysis of isoprene sequences on the core-shell type microspheres was performed in a chloroform solution. Through ozonolysis, the arm chains composed of PS blocks were cleaved at the 1,4-linkage of isoprene units. It was indicated from the dynamic light scattering that the shell thickness on the core-shell type microsphere decreased after ozonolysis. The arm length of PS branching was able to be controlled by ozonolysis at the position of the introduced isoprene units.