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Urease-producing bacteria are highly relevant in medicine due to their role in various pathogenic processes and their impact on human health, causing serious medical conditions such as peptic ulcer disease, gastric cancer, and respiratory and urinary tract infections. In this work, we designed fluorescent polymeric particles (PNP_FITC) to enable the detection of urease-producing bacteria by targeting the enzymatic activity of urease. In particular, the PNP_FITC matrix is degraded by urease, leading to a measurable increase in the intensity of the fluorescent signal. This approach is designed to directly sense urease activity and is therefore not affected by environmental parameters, unlike standard methods based on the quantification of enzymatic metabolites (i.e., NH and CO). PNP_FITC exhibited a linear response in the urease range of 0-7.5 U/mL, with a calculated limit of detection of 0.4 U/mL. The direct detection of enzymatic activity makes PNP_FITC suitable for detecting urease-producing bacteria ( and ) with a detection limit of 10 ∧ 3 bacteria/mL, which were not detectable using the pH-based method employed as the reference in this work. Given the improvements achieved with PNP_FITC in terms of robustness, sensitivity, and selectivity of urease detection compared to the standard methods, this approach represents a step forward toward the development of advanced point-of-care, enabling the prompt diagnosis of bacterial infections.