Elevating the yield and altering the composition of seed tocopherols (Toc’s) are important to rapeseed breeding and production. However, little is known about the biosynthesis of Toc’s in response to environmental signals. In this study, we investigated the effects of exogenous gibberellin (GA3) and paclobutrazol (PAC) on Toc biosynthesis. We also explored the interactive effects between the two plant growth regulators (PGRs) and other factors, such as PGR treatment duration, genotype, and growing location on the total Toc yield and composition in oilseed rape seed. GA3 significantly enhanced the production of Toc’s and elevated the α-/γ-Toc ratio in a time- and genotype-dependent manner. By contrast, PAC significantly reduced Toc yield. Genotypic differences were observed in the effects of GA3 on Toc yield and composition in the seeds. GA3 significantly increased the Toc yield and α-/γ-Toc ratio in Zheyou-50, a genotype with a low proportion of very long chain fatty acids (VLCFAs). However, GA3 did not significantly influence these parameters in Jiu-Er-13Xi, a genotype with a high VLCFA proportion. The increased Toc yield induced by GA3 was mediated by the upregulation of genes (BnPDS1 and BnVTE1) that catalyze the production of Toc precursors. Therefore, applying GA3 can improve rapeseed quality by increasing Toc yield and improving Toc composition.