A new magnetic nanoparticle detection system by using a fundamental mode orthogonal fluxgate (FM-OFG) gradiometer and ac magnetizing coil has been developed. The FM-OFG gradiometer has an active canceling coil on each of its sensor heads against the common magnetic field input to avoid the saturation of the amorphous wire core by a strong ac magnetic field. In addition, the ac magnetizing coil has an adjusting capability to make ac magnetic field strength affecting each of the gradiometer heads equal, which allows us to use a high gain amplifier at the latter stage. Two types of the gradiometer are tested: one is parallel configuration in which two sensor heads are placed in parallel side by side, the other is axial configuration in which two sensor heads are placed axially. Detectable distance was investigated using a 5μL (≈100μg in Fe atomic amount) magnetic nanoparticle sample. The maximum detectable distance for the parallel gradiometer is 17 mm, and that for the axial one is 18 mm.