Objective To make three-dimensional (3D) reconstruction of mouse brain and inner hippcampal formation (HF) with 3D software, and to observe and measure the reconstructed structures. Methods The Nissl stained mouse brain serial cryosections were obtained and pre-processed to construct the image dataset for 3D reconstruction. Using PhotoshopCS3 software, the different anatomic structures of HF in mouse coronary brain sections were fulfilled with different colors according to anatomy knowledge. The 3D-DOCTOR 4.0 software was used to make registration, segmentation and 3D reconstruction of mouse brain and HF, and to observe the reconstructed structures in personal computer (PC). Results The serial Nissl stained cryosections could be used to make 3D reconstruction of mouse HF, and the subiculum, CA1, CA2, CA3, CA4, dentate gyrus (DG), etc, could be observed. Conclusion The serial Nissl stained mouse cryosections could be used to make 3D reconstruction of mouse brain and HF.