A microfluidic thread electroanalytical device (μTED) containing an embedded electrochemical detector is presented for the first time in this work. This novel device was entirely produced in an automated way using the fused deposition modeling (FDM) 3D printing technique. The main platform was fabricated with acrylonitrile butadiene styrene (ABS) filament, while the integrated electrochemical detector was produced using a commercial conductive filament composed of carbon black and polylactic acid (CB/PLA). The microfluidic channels consisted of cotton threads, which act as passive pumps, and the μTED was used for microflow injection analysis (μFIA). As a proof of concept, this μFIA system was utilized for the amperometric sensing of nitrite in natural waters. This is the first report on the use of both μTEDs and 3D-printed CB/PLA electrodes to determine this species. This fully 3D-printed μTED was characterized and all experimental and instrumental parameters related to the method were studied and optimized. Using the best conditions, the proposed approach showed a linear response in the concentration range from 8 to 200 μmol L and a limit of detection (LOD) of 2.39 μmol L. The LOD obtained here was ca. ten-fold lower than the maximum contaminant level for nitrite in drinking water established by the Brazilian and US legislation. Moreover, the platform presented good repeatability and reproducibility (relative standard deviations (RSDs) were 2.1% and 2.5%, respectively). Lastly, the 3D-printed μTED was applied for the quantification of nitrite in well water samples and the results obtained showed good precision (RSD < 3%) and excellent concordance (relative error was ca.±3%) with those achieved by ion chromatography, used for validation.