An on-line sequential injection (SI) system combined with sector field inductively coupled plasma mass spectrometry was developed for the determination of ultratrace level 239Pu and 240Pu in seawater. The potential of this method is the substantial reduction of a sample volume and rapidity in the determination of Pu isotopes. A chemical purification and preconcentration of Pu isotopes were accomplished by the on-line SI system with two microcolumns of solid-phase extraction resins, Sr-Spec and TEVA-Spec. The MCN-6000 microconcentric nebulizer was used as a sample introduction system because of low interference effect and good sample utilization. With this method, it was possible to analyze ultratrace levels of Pu isotopes in only 5 L of surface seawater with an analysis speed of 4 h/sample. The precision of the measurement for the 239Pu and 240Pu was less than 3.4 (n = 7) and 5% (n = 7) for 5 L of seawater. The detection limits for 239Pu and 240Pu were 0.64 (1.5 microBq/mL) and 0.19 fg/mL (1.6 microBq/mL), respectively. The accuracy of this method was verified by using the reference seawater (IAEA-381) as well as by the comparison with the a-spectrometry.