We report an ultrasensitive label-free DNA biosensor with fully on-chip integrated rolled-up nanomembrane electrodes. The hybridization of complementary DNA strands (avian influenza virus subtype H1N1) is selectively detected down to attomolar concentrations, an unprecedented level for miniaturized sensors without amplification. Impedimetric DNA detection with such a rolled-up biosensor shows 4 orders of magnitude sensitivity improvement over its planar counterpart. Furthermore, it is observed that the impedance response of the proposed device is contrary to the expected behavior due to its particular geometry. To further investigate this difference, a thorough model analysis of the measured signal and the electric field calculation is performed, revealing enhanced electron hopping/tunneling along the DNA chains due to an enriched electric field inside the tube. Likewise, conformational changes of DNA might also contribute to this effect. Accordingly, these highly integrated three-dimensional sensors provide a tool to study electrical properties of DNA under versatile experimental conditions and open a new avenue for novel biosensing applications (i.e., for protein, enzyme detection, or monitoring of cell behavior under in vivo like conditions).