DNA 5-hydroxymethylcytosine (5-hmC) is an important epigenetic biomarker for tumorigenesis, and the loss of 5-hmC levels is associated with leukemia and melanoma cancers. However, it is a great challenge to discriminate 5-hmC from 5-methylcytosine (5-mC) using the conventional bisulfite conversion methods. Herein, we report a label-free and immobilization-free electrochemical magnetobiosensor for sensitive quantification of 5-hmC in genomic DNA based on a dual signal amplification strategy coupled with terminal deoxynucleotidyl transferase (TDT) enzymatic amplification and Ru(III) redox cycling. This screen-printed carbon electrode (SPCE)-based electrochemical magnetobiosensor shows distinct advantages of having low cost and simple fabrication and being label-free, immobilization-free, PCR-free, and radioactive-free. It exhibits high sensitivity with a detection limit of as low as 9.06 fM and a large dynamic range from 0.01 to 1000 pM. Importantly, this biosensor can discriminate 5-hmC from cytosine and 5-mC, and it can successfully detect 5-hmC in live cells.