Here we have demonstrated a signal-off architecture for electrochemical aptasensor to detect lysozyme at trace-level. The DNA duplex in which one strand was covalently attached with thiol and the other was tagged with Ferrocene as sensing element. In the absence of target, the DNA duplex was free to collide with the gold electrode surface, the introduction of target lysozyme induced the signal strand to be denatured and released off into the solution. The signals were determined by the square wave voltammograms and were linear with the concentration of lysozyme in the range from 7 nM to 30 nM, with a detection limit of 0.45 nM. The fabricated aptasensor shows a high sensitivity, good selectivity and satisfactory stability. (c) 2013 Elsevier Ltd. All rights reserved.