This work is focused on the optimization of process conditions of the ultrasound-assited extraction that promote high yields of lipids from freeze-dried cells of Mortierella isabellina produced by submerged fermentation. The influence of ultrasound intensity (26.89 W cm(-2) - 85.00 W cm(-2)) and pulse cycle (0.57-1.0) using chloroform:methanol:water (2:1:0.8, v/v/v) mixture and ethanol was evaluated on the yields fatty acids and it was compared with extractions without ultrasound and conventional Soxhlet method. Ultrasound intensity of 75.11 W cm(-2) and pulse factor of 0.93 were the optimized conditions in the central composite design, yielding 14.46 wt% and 19.49 wt% of fatty acids using ethanol and chloroform:methanol:water as solvents, respectively. The fatty acids profile was similar for all extractions. The results show the lipids yields were higher when ultrasound was applied in biomass from fungus Mortierella isabellina.