Magnetic nanoparticle (MNP) agglutination-based optomagnetic biosensing has been applied for rapid and sensitive detection of various biomolecules. However, the underlying physics of optomagnetic biosensing remains a hurdle, especially when the size of MNPs approaches the laser wavelength, i.e., the scattering enters the Mie regime. In this study, we propose a numerical model by combining the Fokker–Planck equation and the T-matrix to explain the effect of Mie scattering on the optomagnetic signal of the agglutinated MNPs. It indicates that the orientation-dependent Mie scattering of the MNP dimer leads to an inverse optomagnetic response compared to that of the MNP monomer. The inverse optomagnetic response can dominate with the enhancement of Mie scattering. To verify the numerical model, biotinylated bovine serum albumin (biotin-BSA) is applied to agglomerate streptavidin-coated MNPs in a standard biosensing strategy representing the immuno-agglutination assay. Guided by the model, a proportion-based signal analysis is proposed to improve the performance of the MNP agglutination-based optomagnetic biosensor, which provides a picomolar detection limit for biotin-BSA (∼0.1 ng/mL) with a total assay time of 8 min, paving the way for more accurate point-of-care diagnosis.