Compounds in Fructus Gardeniae have been shown to possess a wide array of biological activities. However, Gardenia oil extracted from its fruit is less reported and its composition remains uncertain. To completely characterize lipophilic compounds in Gardenia oil, three conventional extraction (CE) and ultrasound-assisted extraction (UAE) methods were investigated. The oil extraction yield obtained by UAE was 51.8% higher than that acquired by cold-pressed extraction (CPE). The fatty acid profile in UAE oil with different solvents was characterized by GC-MS. Petroleum ether was observed to be an ideal solvent with 8.59% extraction yield and 78.88% recovery rate and with a ratio of 3.11 of unsaturated fatty acids to saturated fatty acids. Response surface methodology (RSM) with Box-Behnken Design (BBD) was applied to optimize conditions in UEA of oil to maximize extraction yield. Furthermore, the bioactive components in oil extracted by UAE were qualitatively identified and quantified by HPLC-DAD/ESI-MS(2) and HPLC-DAD analysis. The eight compounds in Gardenia oil, including geniposide, trans/cis-crocin-1, crocin-2, crocin-3, crocin-4, and trans/cis-crocetin, were structurally revealed. The corresponding transfer rates of the bioactive components showed that the lipophilic trans/cis-crocetin could be completely transferred from fruit to oil, with the highest concentration of 11.38 μg g(-1) oil among all compounds quantified. These findings could deliver potential application to a large-scale production of functional Gardenia oil whose bioactive components possess health benefits.