Combining magnetic particle spectroscopy (MPS) with functionalized magnetic nanoparticles (MNPs) has the potential to provide a rapid, sensitive, and user-friendly point-of-care (PoC) testing platform. Conventional MPS techniques typically apply harmonic ratios (such as, the third to fifth harmonic ratio) as the sensing metric to eliminate interference caused by MNP concentration. However, the quantification accuracy of this approach largely depends on the consistency of the signal-to-noise ratio (SNR) between the selected harmonics, imposing stringent requirements on hardware design and the nonlinear magnetic behavior of the MNPs. In this study, we introduce “peak shift” to field-swept MPS (FS-MPS) as a novel metric for MNP characterization and biosensing. An integrated FS-MPS device is used to capture the second harmonic spectrum, which records the second harmonic amplitudes under different perpendicular static magnetic fields. Importantly, the peak position of the spectrum remains constant under different MNP concentrations and shifts upon changes in MNP hydrodynamic size. In this way, the peak shift sensitively detects MNP binding to the targeted analytes, enabling robust biosensing based solely on the second harmonics. The feasibility of the proposed method was demonstrated using streptavidin-grafted MNPs and biotinylated polystyrene beads. Diagnostic performance was assessed using ACE2 functionalized MNPs and SARS-CoV-2 S1 protein and SARS-CoV-2 pseudovirus assays. The proposed strategy can detect virus concentrations as low as 350 TCID50/mL. Additionally, a good linear correlation exists between the peak shift and virus titer in the 350–7000 TCID50/mL range, demonstrating the potential of our method to quantify virus content.