Hydrogen sulfide (H₂S) is the most recently identified endogenous gasotransmitter, along with molecules such as nitric oxide (NO) and carbon monoxide (CO). It has been recognized as a key mediator in various physiological processes, such as cellular homeostasis, as well as in pathological conditions, including cancer. Specifically, H₂S exerts different effects on cancer cells depending on its concentration and exposure time. At lower concentrations, it can promote tumor cell growth and proliferation, whereas higher concentrations may inhibit growth or induce apoptosis. Due to its biphasic behavior, H₂S is a promising biomarker for both diagnostic and therapeutic strategies in cancer, with potential applications in liquid biopsy for non-invasive early detection of tumor progression and treatment response. Given the challenging nature of H₂S detection, there is a growing need for sensitive analytical techniques, such as electrochemical methods, to accurately measure its levels. Considering this, a novel paper-based electrochemical sensor was fabricated for the detection of this gas trasmitter in H₂S-exposed murine tissue lysates, with possible applications in liquid biopsy. The sensor, screen-printed on filter paper, was modified with a Prussian blue (PB) dispersion, synthesized directly on the paper support. Analytical characterization was performed in standard solutions, achieving a detection limit of 3 μM with an adequate repeatability, under 10%. This electroanalytical method was then applied for the detection of H₂S levels in murine skin lysate, untreated cancer murine lysates, and pharmacologically-treated neoplastic murine lysate, validating the accuracy and reliability of the technique. It indicates a notable improvement in portable point-of-care (POC) platforms for cancer diagnostics, allowing rapid, on-site testing with minimal sample volume requirements, simplified procedures, and timely, minimally invasive cancer detection. The use of miniaturized, portable sensors, in particular, allows for a reduction in sample size, which usually presents an issue in traditional approaches.