Ultrasound-assisted extraction (UAE) has attracted growing interest, as it is an effective method for the rapid extraction of bioactive compounds from plants with a high extraction efficiency comparable to the conventional extraction. In this study, UAE was used for the extraction of polyphenols from lipid extracted microalgae (Tetraselmis KCTC 12236BP) and the effects of five extraction variables on the total phenolic compounds (TPC) were studied. For the optimization of extraction parameters, particle size, solid-to-liquid (L/S) ratio, ethanol concentration, extraction temperature and extraction time have been examined as independent variables. All variables exhibited the significant effects on the extraction of TPC and extraction temperature showed the most significant effect among five variables. The optimal extraction conditions were the extraction using mixed particle, S/L ratio of 10%, ethanol concentration of 60%, extraction temperature of 100 °C and extraction time of 30 min, which gave the 8.7 mg GAE/g DW for TPC. Compared with conventional hot-water extraction, TPC extraction under UAE was increased by up to 1.8 fold with same extraction condition. This study showed that UAE under low temperature and short extraction time was proven to be an effective extraction process for TPC production from LEA compared to conventional hot-water extraction process.