Background: As a widely used biomechanical research method, finite element analysis (FEA) is a significant tool for investigating the pathogenesis of disc degenerative diseases and optimizing of spine surgical methods. However, the definitions of the relative nucleus position and its cross-sectional area ratio do not conform to a uniform standard, thus affecting the accuracy (ACC) of the FEA. Hence, this study aimed to determine a precise definition of the relative nucleus position and its cross-sectional area ratio to increase ACC of following FEA studies. Methods: The lumbar relative nucleus position and its cross-sectional area ratio were measured from magnetic resonance imaging data, and then calibrated and validated via FEA. Imaging data from patients without disc degeneration were recruited. The L4-L5 nucleus and disc cross-sectional areas and the distances between the edges of the annulus and nucleus were measured; the ratios between these values were calculated as P1 and P2, respectively. The FEA model was constructed using these measured values, and the relative nucleus position was calibrated by estimating the differences in the range of motions (ROMs) between the model, wherein the ligaments, facet joints and nucleus were supressed, and an in vitro study. Then, ACC were re-estimated in the model with all non-bony structures to validate the measured and calibrated indicators. Results: The interobserver homogeneity is acceptable, and the measured P1 and P2 values are 1.22 and 38%, respectively. Furthermore, an ACC of up to 99% was attained for the model under flexion–extension conditions when the calibrated P1 value (1.62) was used, with a model validation of greater than 90% attained under all loading conditions. Conclusion: The measured and calibrated relative nucleus position and its cross-sectional area ratio increase the ACC of the FEA model, and can therefore be used in subsequent studies.