This study reports on a wash-free, inexpensive and sensitive approach of biomolecule imaging with magnetic nanoparticles (MNPs) via a custom-built scanning magnetic particle spectrometer (SMPS). Streptavidin-coated MNPs are used as magnetic biomarkers for the detection of Immunoglobulin G (IgG) conjugated with biotin (IgG-Biotin) while five samples with different-concentration IgG-Biotin are prepared for experiments. The measurements of the ac susceptibility indicate that the conjugation of the IgG-Biotin onto the surface of the MNPs forms cross-linking between the MNPs, thus increasing the characteristic Brownian relaxation time from 0.627 to 1.448 ms. The ratio of the 3rd to the 1st harmonics is measured on the samples with different-concentration IgG-Biotin in ac magnetic fields with a frequency ranging from about 300 Hz to 2 kHz. It shows that the measurement sensitivity of the IgG-Biotin concentration decreases from 4.62 × 10-3 to 0.39 × 10-3 nM-1 with increasing excitation frequency. Phantom images of the harmonic ratio, measured with the SMPS, indicate that unbound and bound MNPs can be easily distinguished. Furthermore, the excitation frequency dependence of the contrast-to-noise ratio of the images is discussed based on the measurement sensitivity and the standard deviation of the measured image intensity. This study demonstrates the feasibility of the SMPS for imaging biomolecules bound onto the MNPs, which is of great interest to disease diagnostics and therapy.