In this work, an on-line ultrasonic assisted dispersive liquid–liquid microextraction technique using hydrophobic deep eutectic solvent was developed for the first time. The method was successfully coupled with inductively coupled plasma optical emission spectrometer (ICP-OES) for on-line preconcentration and determination of low levels of cobalt in aqueous samples. In this method, micro droplets of dispersed hydrophobic deep eutectic solvent were passed through a membrane filter and then, cobalt ions were eluted directly into the ICP-OES nebulizer. The effects of operational parameters were investigated. Under optimal pH of 5.0, chelating reagent concentration of 2.0 × 10–5 mol L−1, extraction solvent volume of 20 µL, sonication time of 2 mim and eluent flow rate of 4 mL min−1, the detection limit and the linearity of the method were achieved in the ranges of 0.33–200 and 0.09 µg L−1, respectively. Relative standard deviation (%RSD) and enrichment factor of method were 2.8% and 60, respectively. Finally, the proposed method has been successfully applied as an environmentally compatible, reliable, cheap, fast and facile method to the analysis of cobalt in water samples with relative recovery of 90–108%.