Sample preconcentration is an important step to increase the accuracy of further detection, especially for the sample with extreme low concentration. Due to the overlapping of the electrical double layers in the nanochannel, the concentration polarization effect could be generated while applying an electric field. Therefore, a nonlinear electrokinetic flow is induced, which results in fast accumulation of proteins in front of the induced ionic depletion zone, so-called exclusion-enrichment effect. The main purpose of this work is to create nanofissures to achieve the preconcentration of proteins by the exclusion-enrichment effect. The sample of protein is driven by electroosmotic flow and accumulates at the specific location. In this study, the preconcentration chip for proteins was mainly fabricated by simple standard soft lithography with replica of polydimethylsiloxane (PDMS) and fast nanofissures formation by utilizing nanoparticle-facilitaed electric breakdown phenomenon. A novel strategy of nanofissures formation utilizing nanoparticles deposition at the junction gap between microchannels was proposed and dramatically decreased the required electric breakdown voltage in this study. The experimental results indicated that the sample of protein with extreme low concentration of 1 nM was concentrated to 1.5×104-fold in 60 min by the proposed chip herein.