To clarify the extraction conditions of total flavonoids from Perilla frutescens seeds and the content of flavonoids in Perilla frutescens seeds from different regions, ethanol heating extraction and ultrasonic assisted extraction were used to study the extraction concentration, extraction time and extraction temperature. In order to determine the contents of rutin and luteolin in Perilla frutescens seeds from different regions, high performance liquid chromatography (HPLC) was used. The results showed that the total flavonoids from Perilla frutescens seeds was extracted with 80% ethanol, the extraction rate was up to 4.39% with the temperature being controlled at 70℃ and extracting for 120 min. Under the same condition, the extraction rate was up to 4.27% with ultrasound-assisted extracting for 90 min. The ultrasonic extraction method was simple, rapid and accurate, whereas not suitable for long-term extraction. The total flavonoid content in Perilla frutescens seeds from the four tested regions was significantly different (F=17.592?0, P=0.000?7). The content of total flavonoids from Zhongning Ningxia was 1.73 times higher than that from Lantian Shaanxi. And the content of rutin and luteolin in seeds from different regions was different. The content of rutin from Zhongning Ningxia was the highest. The content of luteolin from Duyun Guizhou was the highest.