In this paper, ultrasound-assisted extraction of single cell oil (SCO) from Mortierella isabellina (MI) was investigated using mathematical statistics, such as Plackett-Burman (PB) design, steepest ascent (SA) design, and Box-Behnken (BB) design. The results from response surface methodology (RSM) indicated the hydrochloric acid concentration, ultrasound time, ultrasound temperature, and extraction time are the most significant parameters. The optimum extraction conditions were as follows: ultrasound power 300W, ultrasound time 12.20min, ultrasound temperature 53.42 degrees c, hydrochloric acid 30mmol in each gram of wet fungal mycelia, extraction time 19.45min, extraction solvent (CH3Cl-CH3OH) ratio 2:1. Under the conditions, the extraction rate of SCO from MI was up to 90.63 +/- 1.35%, and the yield of SCO was 109.88 +/- 0.02mg/g (P<0.05) that was 1.23-fold and 1.35-fold the yield of acid-heating method and ultrasound method, respectively.