Objective- To determine the possibility of three-dimensional ultrasonography (3DUS) and measurement of optical nerve sheet by this method. Design- Descriptive study. Animals- 10 mixed normal dogs (5males, 5 females), age 1-1.5 year, and weighting 15±5 kg Procedure- 3D ultrasounds of the eyes were evaluated and the normal optical nerves in 3DUS images were measured using Volouson 730 pro. Results- In the obtained 3D images vitreous body, anterior chamber, and lens cortex and nucleus showed a distinct anechogenic to hypoechogenic. Details of the eyes compartments were better observed by rotating the images in all possible angles and planes using 3D facilities. Anterior and posterior lens capsule and the optic disk were hyperechogenic. The mean optical nerve in males was: 3.85 mm and in females it was 3.86mm. There weren’t a significant difference between ocular nerve measurements of male and female dogs and left and right eyes. Conclusion and Clinical Relevance- The 3DUS gives useful images for teaching and diagnostic purpose and Lesions of the caudal portion of the orbit (e.g. optic nerve atrophy) are better visualized by this technique. The results of the eye 3DU in dogs showed marked advantages in image acquisition for interpretation of all aspects of the ocular structures. Measurement of the optic nerve by 3D ultrasonography and other methods such as CT scan and direct measurement did not have any significant difference.