The hydrodynamic size distribution of magnetic nanoparticle (MNP) clusters is crucial for determining the amounts of biomolecules by analyzing the distributions of the Brownian relaxation time in the corresponding alternating current (AC) magnetization spectra. This study proposes a method for estimating the distribution of MNP clusters from recorded AC magnetization spectra using an experimental-based model. The proposed model is used to form a matrix equation that comprises the distribution and AC magnetization spectra of MNP clusters, and the MNP–biomolecule cluster distribution is obtained by solving an inverse problem. The results show that the variations in the MNP clusters distribution estimated from the model correspond to those measured by dynamic light scattering. The MNP cluster distributions estimated using the experimental model were used to estimate streptavidin concentrations in the range of 0.19–3.38 μM. The estimated values agree accurately with the true values, demonstrating the feasibility of the proposed method for biosensing.