Botulinum neurotoxins are among the most potent toxic bacterial proteins for humans and there is a great need to develop simple, rapid and sensitive methods for toxin detection and protease activity quantification in field deployment. In this paper, a nanoporous membrane based impedance sensor was developed to monitor the activity of the BoNT serotype A light chain protease (LcA). Synaptosomal-associated protein 25 (SNAP-25) was first immobilized inside nanopore walls via silane linkers. BoNT LcA was then injected over the nanoporous membrane substrate sensor and specifically cleaved SNAP-25. The cleavage activity could be monitored by measuring impedance signals across nanoporous membranes which represented the nanopore blockage degree. This initial device could achieve a 500 pM LcA detection limit within 25 minutes.