Noninvasive monitoring of magnetic nanoparticles (MNPs) is gaining increasing importance in the biomedical field. Applications such as drug delivery, contrast imaging, and protein detection require the development of precise and robust experimental setups that closely replicate realistic and physiological conditions. In this study, we investigate the detection and quantification of MNPs under dynamic flow conditions using an unshielded and portable gradiometer setup designed for potential in vivo applications. With its large possible detection area, the setup provides a versatile and adaptable platform for evaluating MNP behavior also in physiologically relevant environments, offering strong potential for translation into diverse in vivo magnetic sensing applications. For testing, MNPs are moved as a bolus through a plastic tube in the sensing area of the setup. An ac susceptibility technique was used to excite the MNPs using a weak alternating magnetic field of 100 µT, while the resulting low MNPs’ magnetization induced a signal detected by two pick-up coils arranged in a differential configuration on one side of the sensing area. Through appropriate calibration of the setup, the magnetic moment of each sample was estimated. The resulting magnetic field was calculated as a function of the sample’s “depth,” i.e., distance from the first coil. For MNPs’ bolus velocities of 10 mm/s, the experimental curves follow the theoretical trend derived from the Biot–Savart law for the magnetic field generated by a single magnetic dipole. 50 µg MNP droplets are detected with a magnitude of 4 nT and an SNR of 8 dB at a distance of 10 mm. The gradiometer sensor is capable of quantifying a 500 µg MNP sample at depths up to 24 mm.