This study develops a novel fluorometric method for the sensitive and selective determination of urea, based on unique system comprising nitrogen doped red-emissive carbon dots (NRECDs), zinc-dithizone complex, and the urease enzyme. The underlying principle of this method relies on the pH increase resulting from the enzymatic breakdown of urea by urease. Initially, the fluorescence of the NRECDs is quenched by the red-colored zinc-dithizone complex. However, upon the addition of urea, the subsequent release of ammonia and the consequent rise in pH lead to the dissociation of the zinc-dithizone complex, causing a color change from red to yellow. This spectral shift eliminates the quenching effect, resulting in the restoration of the CDs’ fluorescence. The prepared NRECDs were comprehensively characterized using various spectroscopic techniques, including fluorometry, X-ray diffraction (XRD), X-ray