Demineralization has been the common method to obtain dentin discs with opened tubules as sensitive teeth defects used for in vitro and in situ dental studies. Bovine teeth have been widely proposed for dental research to overcome the problem of human tooth availability. This study aimed to investigate the morphological changes of demineralized bovine dentin in the fabrication process using HCl 36% to generate the exposal dentin tubule. Scanning electron microscopy (SEM) was used to observe the surface of dentin specimens in each fabrication step. The dentin discs were also fractured into two equal parts for cross-sectional observations. The first 2 h of HCl 36% treatment were effective in eliminating the enamel layer of raw bovine teeth without damaging the inner dentin layer. However, after cutting and shaping, the dentin specimens with pre-etching with HC 36% for 10 s had collapsed dentin walls and dissimilar dentin tubule sizes on both sides of the specimens. The specimens without further pre-treatment maintained their dentin network with smaller dentin tubule sizes. The study suggests developing other simple protocols for making demineralized bovine dentin.