The development of aptamer-based viability impedimetric sensor for viruses (AptaVISens-V) is presented. Highly specific DNA aptamers to intact vaccinia virus were selected using cell-SELEX technique and integrated into impedimetric sensors via self-assembly onto a gold microelectrode. Remarkably, this aptasensor is highly selective and can successfully detect viable vaccinia virus particles (down to 60 virions in a microliter) and distinguish them from nonviable viruses in a label-free electrochemical assay format. It also opens a new venue for the development of a variety of viability sensors for detection of many microorganisms and spores.