Preconcentration of biomolecules by electrokinetic trapping at the nano/microfluidic interface has been extensively studied due to its significant efficiency. Conventionally, sample preconcentration takes place in continuous flow and therefore suffers from diffusion and dispersion. Encapsulation of the preconcentrated sample into isolated droplets offers a superior way to preserve the sample concentration for further analysis. Nevertheless, the rationale for an optimal design to obviate the sample dilution prior to encapsulation is still lacking. Herein, we propose a pressure-assisted strategy for positioning the concentrated sample plug directly at the ejecting nozzle, which greatly eliminates the concentration decline during sample ejection. A distinctive mechanism for this plug localization was elucidated by two-dimensional numerical simulations. Based on the simulation results, we developed an on-demand nanofluidic concentrator in which the nanochannels were facilely generated through lithography-free nanocracking on a polystyrene substrate. By wisely implementing an on-demand droplet generation module, our system can adaptively encapsulate the highly concentrated sample and effectively enhance the long-term stability. We experimentally demonstrated the preconcentration of a fluorescently labelled biomolecule, bovine serum albumin (BSA), by using an amplification factor of 10(4). We showed that, by adjusting the applied voltage, accumulation time, and pulsed pressure imposed on the control microchannel, our system can generate a droplet of the desired volume with a target sample concentration at a prescribed time. This study not only provides insights into the previously unidentified role of assisted pressure in sample positioning, but also offers an avenue for varied requirements in low-abundance biomolecule detection and analysis.