A Nafion®-modified Pt sensor developed for real-time neurochemical monitoring in freely-moving animals has now been characterised in vivo for the detection of nitric oxide (NO). Experiments were undertaken to test sensitivity, selectivity and stability. In control experiments, local administration of aCSF resulted in a decrease in signal (42 ± 12 pA, n = 9), while systemic administration of saline caused a transient increase in signal (22 ± 3 pA, n = 9) from baseline levels. Local MD perfusions of known concentrations of NO (1 mM) and L-arginine (720 μM) caused significant increases in current (4 ± 1 nA (n = 7) and 271 ± 37 pA (n = 5) respectively) while systemic administration of the non-selective NOS inhibitor L-NAME produced a significant decrease in current (91 ± 19 pA, n = 4) compared to baseline levels. Confirmation that the sensor remained selective was achieved by injection of ascorbic acid (2 g kg−1, i.p). The oxidation current showed no variation over the selected time interval of 60 min, indicating no deterioration of the polymer membrane. Finally, after an initial decrease in sensitivity (ca. 50%) within the first 24 h of implantation the Nafion®-modified Pt sensor showed excellent stability under physiological conditions over extended periods (8 days) validating its use for continuous chronic in vivo recordings.