A preconcentration approach for CE-MS using counterflow-assisted electrokinetic injection was proposed. The proposed preconcentration method was based on a counterflow-compatible sheathless interface. The interface was fabricated using a capillary-assembled PDMS microdevice that allowed the application of a counterflow and provided liquid-film electrical conduction. During electrokinetic injection, a hydrodynamic counterflow was introduced into the separation capillary to retard the movement of the stacking boundary to the CE outlet. Accordingly, a long injection time was achieved without a loss of CE separation. With the use of reverse polarity mode and a dynamic polybrene-coated separation capillary (filled with a 1% formic acid solution), a counterflow-assisted electrokinetic injection of peptide samples (in a aqueous solution containing 1% ammonia and 50% methanol) for 10 min provided a sensitivity enhancement of 750-1480 and a detection limit of 20 pM for the five peptide standards. The proposed technique was applied to the analysis of low nanomolar concentrations myoglobin tryptic peptides.