The present article describes the development of a microfluidic-enzymatic sensor with electrochemical detection for the quantification of pipemidic acid (PA), which is a synthetic quinolone used as antibacterial agent. This property of the quinolones is associated with their potential to inhibit topoisomerase II (DNA gyrase) of bacteria. PA detection in pharmaceutical samples was based in the use of tyrosinase enzyme [EC 1.14.18.1] that was immobilized on 3-aminopropyl-modified-controlled-pore glass (APCPG) packet in a central channel (CC) of the microfluidic-enzymatic device. This enzyme catalyzes the oxidation of catechol to o-benzoquinone, whose back electrochemical reduction was detected at gold electrode surface at 0 V vs. Ag/AgCl. Thus, when PA was added to the solution, this piperazine-containing compound participates in a Michael addition reaction with o-benzoquinone to form the corresponding amino-quinone derivative, as result of this interaction the peak current obtained for o-benzoquinone reduction decreased proportionally to the increase of the PA concentration. The recovery of PA from four samples ranged from 97.50% to 102.50%. This method could be used to determine the PA concentration in the range 0.02-70 mu M (r = 0.998) with a limit of detection of 18 nM. This method was successfully applied for the analysis of PA in pharmaceutical formulations. (C) 2010 Elsevier B.V. All rights reserved.