© 2021 The Royal Society of Chemistry and the Centre National de la Recherche Scientifique.This work describes a flow injection analysis (FIA) method for a sensitive, selective and fast detection of hydroxylamine (NH2OH) at a multiwalled carbon nanotube/screen-printed carbon electrode (MWCNT/SPCE) modified with an effective redox mediator of bis-neocuproine Cu(ii) complex ([(Cu(Ncp)2]2+). To fabricate the modified electrode ([(Cu(Ncp)2]2+/Nf-MWCNT/SPCE), negatively charged Nafion (Nf) molecules and [(Cu(Ncp)2]2+ complex were consecutively adsorbed onto MWCNT/SPCE through π-π stacking and electrostatic interactions, respectively. Cyclic voltammograms of the modified electrodes displayed an efficient redox pair, attributed to the reversible oxidation of Cu(i)-Ncp to Cu(ii) chelate complexes. Moreover, this redox couple showed superior electrocatalytic activity towards NH2OH oxidation compared to bare SCPE and Nf-MWCNT/SPCE due to the synergistic combination of MWCNT with the redox mediator. The FI amperometric current response towards electrocatalytic oxidation of NH2OH at +0.35 V vs. Ag/AgCl (0.10 M KCl) exhibited two linear dynamic concentration ranges between 0.25 and 100.0 μM with a detection limit of 0.08 μM (3σ) and between 100.0 and 3000.0 μM. The FI amperometric sensor had high precision and stability, with RSD values of 2.7 and 4.8% (n = 3) for intra-day and inter-day repeatability, respectively. The developed method owes its high sensitivity and selectivity to the 2-e- oxidation of NH2OH to N2O catalyzed by [(Cu(Ncp)2]2+ as the redox mediator, with no side reactions, excellent electrode stability and low pH dependence, unlike other similar literature studies. The proposed electrode was also successfully used for the selective determination of NH2OH in two different real sample types (water and pharmaceutical samples) with satisfactory results.