OBJECTIVE: To evaluate clinical value of three-dimensional (3D) computed tomography (CT) reconstruction of the sphenoid sinus separation in localizing sellar floor during endonasal transsphenoidal surgery and determine size and location of sellar floor fenestration. METHODS: There were 51 patients eligible for study inclusion. Preoperative CT scan of the paranasal sinus and CT scan and magnetic resonance imaging of the pituitary gland were obtained. Sphenoid sinus separation was reconstructed using Mimics 15.0 software, and quantity, shape, and orientation were observed and compared with intraoperative data to guide the localization of sellar floor. Anatomic variation of the sphenoid sinus and adjacent structures, tumor and sella turcica morphology, minimal distance between the cavernous segment of the internal carotid artery bilaterally, and shortest distance from the midline were measured. RESULTS: Based on the shape of the sphenoid sinus separation, sellar floor was accurately localized in all cases. Intraoperative sphenoid sinus separation was consistent with preoperative three-dimensional CT reconstruction images. The sellar floor was extremely small in 2 patients, and insufficient fenestration of sellar floor negatively affected tumor resection. Preoperative three-dimensional CT reconstruction is helpful for accurate and rapid localization of sellar floor. CONCLUSIONS: Anatomic variation of sphenoid sinus and adjacent structures, characteristics of tumor and sella, minimum distance between bilateral cavernous segment of the internal carotid artery, and shortest distance from midline are helpful for establishment of individualized sellar floor fenestration.