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To determine urea accurately in clinical samples, food samples, dairy products, and agricultural samples, a new analytical method is required, and non-enzymatic methods are preferred due to their low cost and ease of use. In this study, bitter gourd peel biomass waste is utilized to modify and structurally transform nickel oxide (NiO) nanostructures during the low-temperature aqueous chemical growth method. As a result of the high concentration of phytochemicals, the surface was highly sensitive to urea oxidation under alkaline conditions of 0.1 M NaOH. We investigated the structure and shape of NiO nanostructures using powder X-ray diffraction (XRD) and scanning electron microscopy (SEM). In spite of their flake-like morphology and excellent crystal quality, NiO nanostructures exhibited cubic phases. An investigation of the effects of bitter gourd juice demonstrated that a large volume of juice produced thin flakes measuring 100 to 200 nanometers in diameter. We are able to detect urea concentrations between 1–9 mM with a detection limit of 0.02 mM using our urea sensor. Additionally, the stability, reproducibility, repeatability, and selectivity of the sensor were examined. A variety of real samples, including milk, blood, urine, wheat flour, and curd, were used to test the non-enzymatic urea sensors. These real samples demonstrated the potential of the electrode device for measuring urea in a routine manner. It is noteworthy that bitter gourd contains phytochemicals that are capable of altering surfaces and activating catalytic reactions. In this way, new materials can be developed for a wide range of applications, including biomedicine, energy production, and environmental protection. more...