Immobilization characteristics for single-stranded oligonucleotide-functionalized magnetic beads with nominal sizes of 40, 80, 130, and 250 nm in rolling circle amplified (RCA) DNA coils is investigated by employing complex magnetization measurements, dynamic light scattering and fluorescence microscopy. It was Found that larger beads in a polydisperse bead size distribution more easily immobilize in the RCA DNA coils than do smaller beads. This may be related to a higher oligonucleotide surface coverage for the larger beads. Furthermore, it was Concluded that both bead size and oligonucleotide surface coverage determine whether beads immobilize to give isolated coils with beads or larger clusters of beads and coils. A small bead size and a low oligonucleotide Surface coverage favor the first kind or immobilization behavior, whereas a large bead size and a high oligonucleotide surface coverage favor the other. The present findings Could be Used to optimize both size and SUI-face functionalization of beads employed in substrate-free magnetic biosensors.