Protein kinases play important roles in regulating various cellular processes and may function as potential diagnostic and therapeutic targets for various diseases including cancers. Herein, we construct a phos-tag-directed self-assembled fluorescent magnetobiosensor to simultaneously detect multiple protein kinases with good selectivity and high sensitivity. In the presence of protein kinases (i.e., PKA and Akt1), their substrate peptides (i.e., a FITC-labeled substrate peptide and a Cy5-labeled substrate peptide) are phosphorylated, and are then specifically recognized and captured by a biotinylated phos-tag to generate biotinylated substrate peptides for the assembly of magnetic bead (MB)-peptides-FITC/Cy5 nanostructures. After magnetic separation, the phosphorylated substrate peptides are disassembled from the MB-peptides-FITC/Cy5 nanostructures using deionized water at 80 °C, releasing FITC and Cy5 molecules. The released FITC and Cy5 molecules are detected by steady-state fluorescence measurements, with FITC indicating PKA and Cy5 indicating Akt1. This magnetobiosensor only involves one phos-tag without the requirement of radiolabeling, antibody screening, carboxypeptidase Y (CPY) cleavage, and cumbersome chemical/enzyme reactions. The introduction of magnetic separation can effectively eliminate the interference from complex real samples, generating an extremely low background signal. Moreover, this magnetobiosensor can accurately measure cellular protein kinase activities and screen inhibitors, with great potential for kinase-related biomedical research and therapeutic applications.