Chemiluminescence (CL) sensing has emerged as a powerful analytical technique for detecting biomarkers in biomedical applications due to its high sensitivity, simplicity, and minimal instrumentation requirements. In this study, we present a smartphone-based chemiluminescence sensing platform for detecting hydrogen peroxide (H₂O₂) and glucose, two critical biomarkers in physiological and pathological processes. By leveraging the widespread availability of smartphones equipped with high-resolution cameras, we developed a low-cost and portable sensing system that captures CL signals, which are subsequently analyzed using ImageJ software and a custom Python-based app. A chemiluminescence sensing method for the determination of H2O2 and Glucose (GC) using a smartphone camera and a 3D printed device made in our laboratory is proposed. The novelty of our approach lies in its integration of advanced imaging capabilities with robust computational tools for real-time intensity quantification and accurate correlation with biomarker concentrations. Using luminol as the CL reagent, we demonstrate the sensitive detection of H₂O₂, which is further utilized in an enzymatic cascade reaction to detect glucose via glucose oxidase-mediated production of H₂O₂. The performance of the system is validated in terms of linearity (0.4–0.9 mM range), detection limits (0.108 mM), and reproducibility, highlighting its potential as a point-of-care diagnostic tool. This work not only bridges the gap between advanced chemiluminescence sensing and practical biomedical applications but also sets a precedent for democratizing healthcare diagnostics using accessible, smartphone-based technology. The proposed platform can be extended to detect other biomarkers, making it a versatile tool for clinical and research applications.