Human sweat is intricately linked to human health, and unraveling its secrets necessitates a substantial volume of experimental data. However, conventional sensors fabricated via complex processes such as photolithography offer high detection precision at the expense of prohibitive costs. In this study, we presented a cost-effective and high-performance wearable flexible sweat sensor for real-time monitoring of K⁺ and Na⁺ concentrations in human sweat, fabricated using screen printing technology. Initially, we evaluated the electrical and electrochemical stability of the screen-printed substrate electrodes, which demonstrated good consistency with a variation within 10% of the relative standard deviation (RSD), meeting the requirements for reliable detection of K⁺ and Na⁺ in human sweat. Subsequently, we employed an “ion-electron” transduction layer and an ion-selective membrane to construct the sensors for detecting K⁺ and Na⁺. Comprehensive tests were conducted to assess the sensors’ sensitivity, linearity, repeatability, resistance to interference, and mechanical deformation capabilities. Furthermore, we evaluated their long-term stability during continuous monitoring and storage. The test results confirmed that the sensor’s performance indicators, as mentioned above, met the requirements for analyzing human sweat. In a 10-day continuous and regular monitoring experiment involving volunteers wearing the sensors, a wealth of data revealed a close relationship between K⁺ and Na⁺ concentrations in human sweat and hydration status. Notably, we observed that consistent and regular physical exercise effectively enhanced the body’s resistance to dehydration. These findings provided a solid foundation for conducting extensive experiments and further exploring the intricate relationship between human sweat and overall health. Our research paved a practical and feasible path for future studies in this domain.