A sequential injection analysis (SIA) system with on-line preconcentration technique using a microfluidic chip with an embedded anionic surfactant ion-selective electrode (AS-ISE) as a detector, was developed for the determination of anionic surfactants. Under the SIA system with no on-line preconcentration technique, the AS-ISE in the SIA system showed a linear relationship between peak heights and logarithmic concentrations of AS ion such as dodecylbenzene sulfonate (DBS) ion with a Nernstian slope of 60.4 mV decade −1 in a concentration range from 3.0 × 10 −6 to 1.0 × 10 −3 mol dm. For the determination of trace levels of ASs, on-line preconcentration technique with solid-phase extraction process was incorporated into the SIA system. By using the on-line preconcentration technique in the SIA system, DBS ion in a concentration range from 1.0 × 10 −7 to 3.0 × 10 −6 mol dm −3 was successfully determined. The recovery for DBS ion added to river water samples was ca. 91-97% using on-line preconcentration technique in the SIA system. The result shows that the present SIA system with the on-line preconcentration technique can be applicable to the determination of the level of AS ion in river and tap water samples.