In this work a novel bioassay for mustard agent detection was proposed. The bioassay is based on the capability of these compounds to inhibit the enzyme choline oxidase. The enzymatic activity, which is correlated to the mustard agents, was electrochemically monitored measuring the enzymatic product, hydrogen peroxide, by means of a screen-printed electrode modified with Prussian Blue nanoparticles. Prussian Blue nanoparticles are able to electrocatalyse the hydrogen peroxide concentration reduction at low applied potential (-50 mV vs. Ag/AgCl), thus allowing the detection of the mustard agents with no electrochemical interferences. The suitability of this novel bioassay was tested with the nitrogen mustard simulant bis(2-chloroethyl)amine and the sulfur mustard simulants 2-chloroethyl ethyl sulfide and 2-chloroethyl phenyl sulfide. The bioassay proposed in this work allowed the detection of mustard agent simulants with good sensitivity and fast response, which are excellent premises for the development of a miniaturised sensor well suited for an alarm system in case of terrorist attacks.