This work aims to remove the oil from passion fruit seeds using ethanol as solvent and then to carry out the transesterification of the product from the extraction step (oil + ethanol). The effects of operational variables in the ultrasound-assisted extraction (UAE) were evaluated and traditional extraction was performed for comparison. The extraction product was directed to the reaction step using an enzymatic catalyst. UAE provided oil yield from 12.32 to 21.76%, and the maximum value (73.7% of the traditional extraction yield) was obtained at 60 °C and 50 min using a solvent-to-seed ratio of 4. Oil removal was favored by increases in the investigated variables. g-tocopherol, δ-tocopherol and a high concentration of polyunsaturated fatty acids were identified in the oils. The oil obtained by UAE presented higher phytosterol contents. From the reaction step, samples were obtained with higher concentrations of ethyl esters, in addition to emulsifiers (diglycerides and monoglycerides).