Functionalized magnetic nanoparticles (mNPs) have shown promise in biosensing and other biomedical applications. Here we use functionalized mNPs to develop a highly sensitive, versatile sensing strategy required in practical biological assays and potentially in vivo analysis. We demonstrate a new sensing scheme based on magnetic spectroscopy of nanoparticle Brownian motion (MSB) to quantitatively detect molecular targets. MSB uses the harmonics of oscillating mNPs as a metric for the freedom of rotational motion, thus reflecting the bound state of the mNP. The harmonics can be detected in vivo from nanogram quantities of iron within 5s. Using a streptavidin–biotin binding system, we show that the detection limit of the current MSB technique is lower than 150pM (0.075pmole), which is much more sensitive than previously reported techniques based on mNP detection. Using mNPs conjugated with two anti-thrombin DNA aptamers, we show that thrombin can be detected with high sensitivity (4nM or 2pmole). A DNA–DNA interaction was also investigated. The results demonstrated that sequence selective DNA detection can be achieved with 100pM (0.05pmole) sensitivity. The results of using MSB to sense these interactions, show that the MSB based sensing technique can achieve rapid measurement (within 10s), and is suitable for detecting and quantifying a wide range of biomarkers or analytes. It has the potential to be applied in variety of biomedical applications or diagnostic analyses. •The freedom of rotational motion of mNP can be measured by MSB within seconds.•The MSB signal of mNP decreased with increasing analyte concentration.•We optimized the sensing scheme using streptavidin/biotin as the model system.•MSB can detect protein and DNA with high specificity and sensitivity.•MSB is a versatile technique, suitable for both in vitro and in vivo detection.