Lipid extraction is a key step of biodiesel production from microalgae, however, the application of traditional extraction methods was limited due to the difficulties of cell disruption as well as solvent toxicity. In this work, pretreatment method using pulsed electric field (PEF), was primarily applied to lipid extraction process from microalgae Chlorella pyrenoidosa grown in wastewater. After the pretreatment with PEF, the yields of fatty acid methyl esters from C.pyrenoidosa was 12.0% higher than traditional pretreatment with ultrasonic. The results indicated that PEF was an effective method for cell disruption. Fluorescence staining and scanning electron microscopy showed that the integrity of the cell membrane of microalgae was damaged under pulsed electric field, which enhanced the penetration of solvents and lipid extraction.