In this work, we report the development of a silver-modified gold–gold microtrench electrode for nitrate detection which shows good sensitivity in generator–collector mode within a physiologically relevant concentration range (200–1400 µM). The proposed sensing mechanism is shown in Fig. 1 (the redox system nitrite/nitrate is represented by Red/Ox). Within the microtrench, nitrate is converted by reduction to nitrite at one electrode (the “generator”), while nitrite is converted back to nitrate by oxidation at the other electrode (the “collector”). A repeating – and so signal-amplifying – redox cycle is achieved as these species interconvert and diffuse across the gap between the electrodes. After development of the electrode system in aqueous buffer, the analytical parameters of the electrode were investigated in horse serum diluted in phosphate buffer. The concentration of nitrate present in the samples was determined by the method of standard addition and compared against a standard gas-phase chemiluminescence method using a chemical analyser.