Microbial safety of drinking water constitutes a major concern in countries at all levels of economic development. Thus, rapid, sensitive and cost effective methods of pathogenic bacteria detection, like common like Escherichia coli O157:H7, which can cause important diseases, is highly required. In this work an impedimetric transducer modified with E. coli specific aptamer is studied. To enhance the sensitivity a three-dimensional interdigitated electrode array (3D-IDEA) impedimetric transducer in which the electrodes separated by insulating barriers was used. In this sensor, chemical reactions at the surface of the barrier provoke electrical charge redistribution which causes changes in surface conductivity. A DNA aptamer that recognizes specifically the outer membrane proteins of the E. coli O157:H7 was selected as the biorecognition moiety. Here we report a novel label-free impedance aptasensor for detection and quantification of pathogenic E. coli O157:H7 with a low detection limit, good selectivity and short detection times. The developed sensor shows a linear response (R 2 =0.977), proportional to the logarithm of bacterial concentration present in the sample, with a limit of detection (LOD) of about 10 2 cfu•mL-1. No response was registered when the aptasensor was incubated with other bacterial strains, confirming the selectivity of suggested method. Additionally, the possibility of the sensor regeneration is shown, so that the detection may be performed several times with the same sensor. Moreover, suitability of the aptasensor for bacteria detection in real samples was demonstrated with a new approach involving bacteria pre-concentration.