In this work, we have studied field-induced aggregation and magnetic separation—realized in a microfluidic channel equipped with a single magnetizable micropillar—of multicore iron oxide nanoparticles (IONPs) also called " nanoflowers " of an average size of 27 ± 4 nm and covered by either a citrate or polyethylene (PEG) monolayer having a thickness of 0.2–1 nm and 3.4–7.8 nm, respectively. The thickness of the adsorbed molecular layer is shown to strongly affect the magnetic dipolar coupling parameter because thicker molecular layers result in larger separation distances between nanoparticle metal oxide multicores thus decreasing dipolar magnetic forces between them. This simple geometrical constraint effect leads to the following important features related to the aggregation and magnetic separation processes: (a) Thinner citrate layer on the IONP surface promotes faster and stronger field-induced aggregation resulting in longer and thicker bulk needle-like aggregates as compared to those obtained with a thicker PEG layer; (b) A stronger aggregation of citrated IONPs leads to an enhanced retention capacity of these IONPs by a magnetized micropillar during magnetic separation. However, the capture efficiency Λ at the beginning of the magnetic separation seems to be almost independent of the adsorbed layer thickness. This is explained by the fact that only a small portion of nanoparticles composes bulk aggregates, while the main part of nanoparticles forms chains whose capture efficiency is independent of the adsorbed layer thickness but depends solely on the Mason number Ma. More precisely, the capture efficiency shows a power law trend Λ ∝ Ma −n , with n ≈ 1.4–1.7 at 300 < Ma < 10^4 , in agreement with a new theoretical model. Besides these fundamental issues, the current work shows that the multicore IONPs with a size of about 30 nm have a good potential for use in biomedical sensor applications where an efficient low-field magnetic separation is required. In these applications, the nanoparticle surface design should be carried out in a close feedback with the magnetic separation study in order to find a compromise between biological functionalities of the adsorbed molecular layer and magnetic separation efficiency.