The objective of this study was to evaluate the accuracy of a CT-based, 3D-printed, patient-specific brain biopsy guide (3D-psBBG) through the application of a transfrontal approach in canine cadavers. A total of ten canine cadavers, with weights ranging from 4.36 to 14.4 kg, were subjected to preoperative CT scans to generate 3D skull models. Customized biopsy guides were created based on these models and manufactured using 3D printing technology. Twenty spinal needle insertions were performed, and the accuracy of needle placement was evaluated through both CT and 3D volume-rendering techniques. The mean needle placement error was 2.1 mm, with no significant differences observed between insertions targeting the fronto-olfactory and piriform lobes. The 3D volume-rendering method demonstrated superior accuracy compared to the CT method, with statistically significant differences in placement errors for both targets. The average time required for the design and manufacture of the guides was 249 min. These findings indicate the high accuracy and potential clinical application of CT-based 3D-psBBG for improving diagnostic outcomes in veterinary neurology.