Herein, mannitol capped magnetic nanoparticles were fabricated for dispersive micro-solid-phase extraction of sparfloxacin (SPX) and orbifloxacin (ORX) from milk and natural water samples. The synthesized magnetic sorbent is readily dispersed in the water and easily isolated magnetically from the medium after loaded with the cited analytes. The effect of main adsorption experimental parameters were studied and optimized. The small amount of the sorbent (15 mg) is applicable for the preconcentration in a reasonable time (15 min). Additionally, the adsorption characterization fits the pseudo-second order kinetic and Langmuir isotherm model with a high determination coefficient. Magnetic nanoparticles were modified with mannitol polar groups to adsorb polar drugs (SPX and ORX) from complicated matrices via hydrogen bonding and electrostatic interactions. After micro-extraction, SPX and ORX were analyzed using boron-doped carbon quantum dots (B-CQDs) as a fluorescence nanoprobe, as the extracted SPX and ORX favor the fluorescence of B-CQDs via hydrogen bonding and electron donor-acceptor association (charge-transfer complex). The synthesized nanomaterials were characterized by different techniques such as Scanning Electron Microscope (SEM), Transmission Electron Microscope (TEM), Fourier-transform infrared spectroscopy (FTIR), Powder X-ray diffraction (PXRD), Energy-dispersive X-ray spectroscopy (EDS), and UV/Vis spectroscopy. Under the optimum conditions, the fluorimetric method exhibited the increase of fluorescence intensity in the range of 1.5 × 10−9–1.3 × 10−7 M and 1.2 × 10−9–1.1 × 10−7 M with limits of detection (LODs) of 5.0 × 10−10 and 4.0 × 10−10 M for SPX and ORX, respectively. The proposed dispersive micro-solid-phase extraction based fluorimetric method was applied for the determination of SPX and ORX in milk and natural water samples with satisfactory results.