The volatile compounds responsible for the pleasant aroma of virgin olive oil are mainly derived from products of the lipoxygenase pathway. Lipoxygenase activity was assessed in olive pulp extracts. An optimization of its extraction by a 2 4 experimental design was performed. Four parameters considered as influencing the extraction were optimised using minimum and maximum levels. The polyvinylpolypyrrolidone quantity seemed to be the more influencing parameter, followed by ethylenediamine tetraacetic acid concentration, pH value, and dithiothreitol concentration. To establish whether the levels of lipoxygenase activity correspond to the enhancement of olive oil aroma, we investigated the addition of soybean lipoxygenase extracts during malaxation. Volatile profiles analysis showed that lipoxygenase had a potential effect on the production of green and fruity note of olive oil.