Selective isolation of mono-and multi-phosphorylated peptides is important for understanding how a graded protein kinase or phosphatase signal can precisely modulate the on and off states of signal transduction pathways. Here we report that metal ions at exposed octahedral sites of nano-ferrites, including Fe 3 O 4 , NiFe 2 O 4 , ZnFe 2 O 4 and NiZnFe 2 O 4 , have distinctly selective coordination abilities with mono-and multi-phosphopeptides. Due to their intrinsic magnetic properties and high surface area to volume ratios, these nanoparticles enable the rapid isolation of mono-and multi-phosphopeptides by an external magnetic field. Model phosphoprotein a-casein and two synthesized mono-and di-phosphopeptides have been chosen for proof-of-principle demonstrations, and these nanoparticles have also been applied to phosphoproteome profiling of zebrafish eggs. It is shown that NiZnFe 2 O 4 is highly selective for multi-phosphopeptides. In contrast, Fe 3 O 4 , NiFe 2 O 4 and ZnFe 2 O 4 can bind with both mono-and multi-phosphopeptides with relatively stronger affinity towards monophosphopeptides.