This article describes a thin amperometric nitric oxide (NO) sensor that can be microchannel embedded to enable direct real-time detection of NO produced by cells cultured within the microdevice. A key for achieving the thin (∼1 mm) planar sensor configuration required for sensor-channel integration is the use of gold/indium−tin oxide patterned electrode directly on a porous polymer membrane (pAu/ITO) as the base working electrode. The electrochemically deposited Au−hexacyanoferrate layer on pAu/ITO is used to catalyze NO oxidation to nitrite at lower applied potentials (0.65−0.75 V vs Ag/AgCl) and stabilize current output. Furthermore, use of a gas-permeable membrane to separate internal sensor compartments from the sample phase imparts excellent NO selectivity over common interfering agents (e.g., nitrite, ascorbate, ammonia, etc.) present in culture media and biological fluids. The optimized sensor design reversibly detects NO down to the ∼1 nM level in stirred buffer and <10 nM in flowing buffer when integrated within a polymeric microfluidic device. We demonstrate utility of the channel-embedded sensor by monitoring NO generation from macrophages cultured within non-gas-permeable microchannels, as they are stimulated with endotoxin.