This study explores the application of a tyrosinase cantilever nanobiosensor for detecting 17β-estradiol and estrone in typical water systems. The physical-chemical parameters of water were evaluated within the Tigre River micro-basin in Erechim, RS, to determine water potability for urban populations. Water clarity, conductivity, and pH levels were essential markers, adhering to recognized standards for water quality and human consumption. The cantilever nanobiosensor demonstrated strong sensitivity and a broad linear range, with a limit of detection (<0.00051 ppb) surpassing other enzymatic biosensors and covering a range of 0.0001–100 ppb.The real water sample quality investigated in relation to contamination with 17β-estradiol and estrone by nanobiosensor showed values below the LOD for both compounds. Recovery studies demonstrated the reliability of the nanobiosensor. Selectivity tests indicated minimal interference from structurally similar substances. This study validates the nanobiosensor's potential for environmental monitoring and hormone detection, aligning with standard practices.