An automated method was developed for rapid determination of 99Tc in large volume seawater samples. The analytical procedure involves preconcentration of technetium with coprecipitation, online separation using extraction chromatography (two TEVA columns) implemented in a sequential injection setup, and measurement of 99Tc by inductively coupled plasma mass spectrometry (ICP-MS). Chromatographic behaviors of technetium, molybdenum, and ruthenium were investigated, and the mechanism of adsorption and elution of TcO4– on a TEVA column using HNO3 was explored. The results show that not only NO3– but also acidity (or concentration of H+) of the loading or eluting solution affect the adsorption and desorption of TcO4– on TEVA resin. Decontamination factors of more than 1 × 106 for ruthenium and 5 × 105 for molybdenum are achieved. Chemical yields of technetium in the overall procedure range from 60% to 75% depending on the sample volumes, and a detection limit of 7.5 mBq/m3 (or 11.5 pg/m3) for 200 L of seawater was obtained. Compared with the conventional analytical procedure, the developed method significantly reduces analytical time. A batch of samples (n > 4) can be analyzed within 24 h. The method has been successfully applied for rapid and automated determination of low level 99Tc in large volume seawater samples. The analytical results of seawater samples collected in Denmark show a good agreement with the values obtained using the conventional method.