Nickel oxide nanoparticles were effectively synthesized through a simple, cost-effective and ecologically safe green synthesis approach by using Adhatoda zeylanica leaf extract as a bioreducing agent and Nickel chloride hexahydrate as the metal precursor. The synthesized NiO NPs were thoroughly characterized via UV-V is spectroscopy (band gap energy of 2.83 eV), FT-IR, XRD, SEM and TEM analyses, thereby affirming their formation, morphology and crystallinity. The characterization outcomes exhibited a strong correlation with previously published data. This research focused mainly on the development of a non-toxic and inexpensive electrochemical sensor for the detection of L-tryptophan. A glassy carbon electrode covered with Adhatoda zeylanica-mediated NiO NPs was used for sensing in phosphate buffer solution. Using cyclic and differential pulse voltammetry, the optimum efficiency of the sensor was found at pH 3.0 confirming a pH-dependent, irreversible twoelectron/two-proton oxidation mechanism driven by adsorption and diffusion. In the L-trp concentrations range of 1 to 12 µM, the sensor showed a linear response with low detection and quantification limits of 0.92 μM and 3.06 μM, respectively.