In this work a microflow structure, suitable for μ-FIA (micro flow injection analysis), will be described, evaluated and applied to real samples. Microchannels, the detector flow cell and input/output ports have been micromachined in silicon and sealed with anodically bonded Pyrex glass. The channels are defined by etching approximately 200 µm depth in silicon using a dry reactive ion etching (RIE) process. Optical windows integrated in the chip structure allow simple absorbance/transmission measurements to be made. The optical measurements were made using an LED as emitter (λ = 525 nm) and a photodiode as a detector. A Visual-Basic program has been developed to control an automatic burette, three-way solenoid valves and the data acquisition system. The μ-FIA for nitrite determination using the Griess–Ilosvay reaction has been implemented for the on-line monitoring of wastewater treatment plants (WWTPs). The multicommutation concept has been applied in order to enhance the mixing process inside the microsystem. Tandem streams of reagent and sample were generated and evaluated at different commutation frequencies. Two optimal frequencies, 400/200 ms and 150/450 ms, were found to be the most suitable ones. The first commutation ratio gave rise to wide linear working range (0–250 ppm), in spite of a high detection limit (0.35 ppm) and a low sensitivity (0.0041 ± 0.0004 AU ppm−1). With the second ratio, the working linear range was smaller (0–50 ppm) but the detection limit (0.17 ppm) and the sensitivity (0.0091 ± 0.0003 AU ppm−1) improved remarkably. Finally, real samples with a high nitrite concentration (0–1500 ppm) coming from a study of kinetic inhibition in the nitrification process at a WWTP has been analysed with the proposed μ-FIA system. The obtained results have allowed the corroboration of the model of inhibition by the nitrite ion with great exactitude.