Diabetic foot ulcers (DFUs) are a serious complication of diabetes with rising prevalence. Current diagnostic methods are limited to macroscopic wound analysis. Molecular diagnostics hold the potential to enhance the diagnosis, staging, and evaluation of treatment response for DFUs. In this study, we created a fluorescent pH-sensing bandage for point-of-care wound diagnostics. Through a fluorescent dye screening process, pyranine emerged as the lead compound due to its effective pH-sensing properties within the clinically relevant pH range of 6–9. To minimize the dye's release into the wound bed, we explored a library of ionic microparticles, discovering a robust adsorption of the anionic dye to a cationic polymeric microparticle. These dye-loaded microparticles exhibited a robust fluorescence response in the targeted pH range and a dye release below 1% in biological fluids. They were encapsulated in a highly biocompatible calcium alginate hydrogel to reduce interaction with the wound tissue. The hydrogel underwent testing on full-thickness dorsal wounds in mice, revealing a linear fluorescence response (R2 = 0.9909) to pH values in a clinically relevant range. These results encourage the further development of this pH-sensing wound dressing for molecular diagnostics in DFUs.