The oculomotor nerve (OCN) innervates the eye muscles and can be affected by inflammatory, compressive, and pathological conditions. Diffusion MRI (dMRI) tractography shows the potential ability to describe the trajectory of the OCN. However, reconstruction of the OCN in the cavernous sinus is still challenging due to the complex tissue environment at the skull base.In this study, we integrated anatomical knowledge to propose a unified framework for OCN tractography, using 45 dMRI datasets from the Human Connectome Project subjects aged 22-36 years and data from four neurosurgical patients aged 41-53 years with visual behavior disorders. We first employed automatically labeled direct and indirect anatomical landmarks as reference locations for individualized tractography. Next, we compared five widely used cranial nerve reconstruction algorithms to assess the most suitable method for OCN reconstruction. Finally, we tested the combination of the unified framework and the optimized tractography method in tumor patients.We found that unscented Kalman filter (UKF)-2T and probabilistic tractography outperformed other methods in OCN fiber tractography, owing to their "step-by-step" fiber direction computation and multidirectional consideration, respectively. In neurosurgical patients, UKF-2T effectively reconstructed OCN fibers around lesions.Our study provides valuable insights for researchers and clinicians in the diagnosis and treatment of OCN-related diseases and neurosurgeries.© 2025 American Society of Neuroimaging.