Modern analytical chemistry, enhanced by advancements in materials science, is crucial for cost-effectively improving measurement techniques, and making analyses more accessible and user-friendly. Disposable screen-printed electrodes have emerged as promising tools, offering miniaturization, high reproducibility, high sensitivity, rapid detection, and inherent selectivity. This study highlights the development of advanced 2D Ti3C2Tx MXene-modified disposable screen-printed gold electrodes for non-invasive, enzyme-free glucose sensing. Ti3C2Tx was synthesized using direct and in-situ etchants to achieve controlled surface chemistry. Techniques such as XRD, UV–visible spectroscopy, SEM, and TEM were employed to characterize Ti3C2Tx synthesized with different etchants. The synthesized materials were then used as working electrodes to investigate their electrocatalytic performance, tailored for continuous glucose monitoring. Upon optimization, in-situ LiF and HCl etched Li-Ti3C2Tx@SPGE exhibited a high sensitivity of 569.70 µA mM−1cm−2, followed by HF-Ti3C2Tx@SPGE and N-Ti3C2Tx@SPGE with sensitivities of 249.62 and 297.78 µA mM−1cm−2, respectively. These sensors demonstrated low limits of detection (LOD) of 5 nM, 28 nM, and 19 nM within a linear range of 5 to 350 µM. Additionally, real sweat analysis was conducted by collecting natural sweat through fast walking and evaluating sensor performance after beverage consumption. Overall, Li-Ti3C2Tx@SPGE, without any additional composition or doping, shows potential as a superior commercial sweat sensor for routine glucose monitoring. This study underscores the innovative use of 2D materials in developing highly sensitive, non-invasive sweat glucose sensors that are both effective and accessible.