Dental anatomy is an integrated, core fundamental dental course, which prepares students for all future clinical dental courses. This study aimed to build up an online dental learning platform of micro-computed tomography-based three-dimensional (3D) tooth models with pulp cavity, and to further evaluate its effectiveness for dental anatomy education using a cohort study. First, ninety-six extracted permanent teeth were scanned by micro-computed tomography and the enamel, dentine, and pulp cavity of each was distinguished by different grey-scale intensities using Mimics software. Three-dimensional images allowed further discrimination and insights into permanent three-rooted premolars, central tip, and dental diseases including deep caries and wedge-shaped defects. Furthermore, a second mesiobuccal canal (MB2) in maxillary permanent molar teeth and Vertucci type III root canal configuration in mandibular anterior teeth could be detected using the 3D analytical tool. A digitized 3D tooth model learning platform was implemented. Last, two groups of dental students were assessed to evaluate the effect of 3D models on dental anatomy education. Participants in the Digital group were allowed to use the online dental learning platform freely after class, while the participants in the Traditional group were not. Assessment quizzes showed that participants' scores improved in the Digital group with the use of the learning platform compared with scores in the Traditional group. A questionnaire survey indicated that the participants had a positive attitude toward the 3D models. Thus, adding digital 3D resources to a traditional curriculum may have a positive effect on academic achievements.