NiOx thin films were prepared on an Au substrate using the electrodeposition (ED), spin-coating (SP), and ED after SP (SP/ED) techniques to realize their application as an electrochemical sensor for the selective detection of trace substances. Results indicated that the electrodeposited films had nanoparticles formed as coarse-grain morphology, and the spin-coated films had a uniform layer with ~60 nm thickness. The thin film prepared by the SP/ED technique showed the highest electrochemical activity, and it was used to record a linear sweep voltammogram to measure the target substance, MSG and glucose, from low concentrations (2 nM) to high concentrations (200 μM). Within the range of concentrations, high R2 values of ≥0.99 were observed for both target substances, confirming that the SP/ED thin films can be used as an electrochemical sensor with high reliability.