This study aims to find accurate angles and depths of lateral ventricle puncture using diffusion tensor imaging (DTI) reconstruction, as well as to provide an optimized and alternative puncturing strategy. A total of 90 computed tomography (CT) images and 30 CT images with DTI were analyzed. The measurements were performed on coronal, sagittal, and horizontal planes. Some distances and angles were measured to determine the best angle and penetration depth during the puncture process. Important landmarks of the lateral ventricle were also measured, and a comparison of the differences between 2 hemispheres was also assessed. It showed that the vertical distance from the superior margin to inferior margin of the lateral ventricle was 22.2 +/- 0.5mm and the length was 124.1 +/- 2.1mm. In the frontal horn puncture approach, the penetration depth should be limited between 105.2 and 109.4 mm, the angle should be 71.6 +/- 2.7 degrees. During the occipital horn puncture approach, puncturing depth was from 90.7 to 111.4mm, and angle was 15.3 +/- 1.8 degrees. Through the parietal lobe puncture approach, which was firstly brought out in this study, the puncturing length should be 124.4 to 130.2 mm and angle was 56.6 +/- 2.0 degrees. The traditional recommended protocol of lateral ventricle puncture is not accurate, the refined lateral ventricle puncture protocol established in this study will reduce injury and remain function. A DTI imaging examination combining with nerve fibers reconstruction were strongly recommended before lateral ventricle puncture, which will help neurosurgeons to determine the best puncturing angles and depth.