The development of traditional enzyme-linked immunosorbent assay (ELISA) systems for the detection of small-molecule residues in foods is limited because of the poor stability of biological enzymes and the lower sensitivity of absorption-based signals. Herein, two ELISAs based on Prussian blue nanoparticles (PBNPs) were developed to establish ultrasensitive and stable methods for detecting norfloxacin (NOR) in milk. The results show that the detection limit (IC15) of NOR was 0.77 μg L−1 and the sensitivity (IC50) was 18.28 μg L−1 in the standard solution using the PBNPs-based nano-ELISA. When the fluorescence quenching ELISA based on PBNPs was used, the detection limit was 0.06 μg L−1 and the sensitivity was 4.21 μg L−1 in the standard solution. The recoveries and precision were good, as confirmed by analysis of real milk samples. The results were consistent with those of commercial ELISA kits, indicating the high accuracy of these two methods.