Pyoverdine is a fluorescent siderophore produced by Pseudomonas aeruginosa that can be considered as a detectable marker in nosocomial infections. The presence of pyoverdine in water can be directly linked to the presence of the P. aeruginosa, thus being a nontoxic and low-cost marker for the detection of biological contamination. A novel platform was developed and applied for the electrochemical selective and sensitive detection of pyoverdine, based on a graphene/graphite-modified screen-printed electrode (SPE) that was electrochemically reduced and decorated with gold nanoparticles (NPs). The optimized sensor presenting higher sensitivity towards pyoverdine was successfully applied for its detection in real samples (serum, saliva, and tap water), in the presence of various interfering species. The excellent analytical performances underline the premises for an early diagnosis kit of bacterial infections based on electrochemical sensors.