In this study, a real-time detection method for amol biomolecules based on second and third harmonics of water-based magnetic nanoparticles (MNPs) is reported. In previous magnetic immunoassay methods based on magnetic particle spectroscopy (MPS), the first harmonic was affected by the diamagnetic environment and would easily interfere with the signal at a concentration lower than pM MNPs. We propose to use the ratio of the third to second harmonics to achieve more sensitive immune detection by using an ac plus dc excitation magnetic field. We found that a homemade magnetic particle spectrometer can ensure that the signal-to-noise ratios (SNRs) of the second and third harmonics are roughly the same by modulating the dc magnetic field to compare and adjust the amplitudes of the second and third harmonics. The immunoassay sensitivity was greatly improved, and the ability of magnetic immunoassay based on water-based MNPs to detect trace biomolecules was roughly estimated using the limit of detection (LOD) for 0.4 pM (40 amol) biomolecules in a 100- suspension, by binding streptavidin to biotin. In addition, the entire test is completed within 1 s, which will be useful in testing for viral aerosols in the air.