AbstractTinnitus, characterized by an auditory perception in the absence of actual sound, is one of the most common encountered difficulties to manage otological problems, in which, lidocaine has been shown to provide temporary relief from subjective tinnitus. However, the pathophysiological mechanism of lidocaine treatment remains to be understood. The study demonstrates the first observation on that lidocaine well suppresses the decrease in the level magnesium (Mg2+) in inferior colliculus (IC) of guinea pigs following salicylate‐induced tinnitus with an online electrochemical system (OECS) for continuously monitoring of microdialysate of Mg2+. The OECS was established by efficiently coupling a selective electrochemical detector with in vivo microdialysis to monitor Mg2+ in the microdialysate continuously sampled from animal brain. The selective detection of Mg2+ over other coexisting neurochemicals was successfully achieved through a mechanism with divalent metal cations (e. g., Mg2+, Ca2+) enhanced catalytic current of organic dyes (i. e., polymerized film of toluidine blue O, p‐TBO) toward the oxidation of nicotinamide adenine dinucleotide (NADH) and with ethyleneglcol‐bis (2‐aminoethylether) tetraacetic acid (EGTA) as selective masking agents for Ca2+. Tinnitus was induced by intraperitoneal injection of sodium salicylate (350 mg/kg body weight). For the lidocaine group, using the microdialysis probe, 1 % lidocaine was perfused into IC of guinea pigs at 2 μL/min for 20 min. With the OECS, we found that the perfusion of lidocaine could recover the decrease of the Mg2+ level caused by salicylate‐induced tinnitus. The basal level of microdialysate Mg2+ in IC of guinea pigs was previously determined to be 1.29±0.44 mM, which was decreased significantly following salicylate‐induced tinnitus. The lidocaine perfusion significantly recovers the Mg2+ level by 37.7±14.8 %. This observation suggests that Mg2+ plays an important role in the pathological mechanism of tinnitus.