Enhanced sweat sensors lead to real-time, sustained, noninvasive tracking of sweat loss, ensure insight into individual health conditions at the molecular level, and have obtained prominent interest for their hopeful implementations in customized health tracking. Metal-oxide-based nanostructured electrochemical amperometric sensing materials are the best selection for continuous sweat monitoring devices owing to their high stability, high-sensing capacity, cost-effectiveness, miniaturization, and wide applicability. In this research, CuO thin films have been fabricated by successive ionic layer adsorption and reaction technique (SILAR) with and without the addition of Lawsonia inermis L. (Henna, (LiL)) leaf extract (C10H6O3, 2-hydroxy-1,4-naphthoquinone) with a high-sensitive and rapid response for sweat solution. Despite the pristine film being responsive to the 65.50 mM sweat solution (S = 2.66), the response characteristic improves to 3.95 for the 1.0% LiL-implemented CuO film. Unmodified, 1.0% LiL and 3.0% LiL-substituted thin-film materials assure considerable linearity with linear regression ranges, R2, of 0.989, 0.997, and 0.998, respectively. It is noteworthy here that this research aims to determine an enhanced system that could potentially be implemented in real-life sweat-tracking administrations. Real-time sweat loss tracking capabilities of CuO samples was found to be promising. Derived from these outcomes, we concluded that the fabricated nanostructured CuO-based sensing system is a useful application for the continuous observation of sweat loss as a biological argument and compatibility with other microelectronic technologies.