Oil-tea (), a typical oilseed tree, produces high-quality edible vegetable oils that contain rich unsaturated fatty acids and diverse lipid-soluble active compounds such as squalene. Although squalene biosynthesis and its molecular regulation have been studied in several plants, the molecular mechanisms underlying squalene biosynthesis in oil-tea seeds remain uncertain. We investigated and determined squalene accumulation with seed development. We conducted comparative transcriptomic analyses using the RNA-seq technique at the early, fast biosynthesis, and late stages of squalene accumulation with oil-tea seed development and identified 13 squalene biosynthesis key enzyme genes (such as , , , and ) in developing oil-tea seeds. According to whether the expressions of key enzyme genes were associated with squalene accumulation we found that the precursor IPP of squalene biosynthesis obtained via the MVA pathway was dominant with oil-tea seed development. Based on the gene co-expression analyses, we identified multiple transcription factors potentially involved in regulating squalene biosynthesis such as , , , and . Using yeast one-hybrid and dual-luciferase assay experiments we demonstrated that the transcription factor could activate the expression of a key enzyme gene , suggesting that might be a critical regulator during squalene biosynthesis in oil-tea seed development. This study gives not only insights into understanding the molecular basis of squalene biosynthesis in oil-tea developing seeds but also provides gene resources for developing genetically improved varieties with higher content of squalene in oil-tea.