The present study was undertaken to separate the available components effectively, such as tetrodotoxin(TTX), docosahexaenoic acid(DHA, C22:6,ω-3) and eicosapentaenoic acid (EPA, C20:5,ω -3) from pufferfish liver waste, which are known to have high values as bioactive materials. By using ultrafiltration, it was possible to separate high contents of 68mg TTX from pufferfish liver waste. In contrast, by activated charcoal column, it was to obtain about 54mg TTX. The recovering ratios were 65.3% and 45.0% in the two different methods of ultrafiltration and activated charcoal column, respectively. From the results of HPLC and gas chromatography-mass spectrometry(GC-MS), the obtained toxins were identified to be TTX and its derivatives. In addition, it was also possible to obtain 72.3g DHA and 11.4g EPA from 1kg of pufferfish liver by high performance liquid chromatography (HPLC). These amounts of DHA and EPA were also 17.70% and 1.04% in the total lipid of pufferfish liver oil from analysis of gas chromatography(GC), respectively.