Herein, we applied hypericin (Hyp) for the detection of DNA on streptavidin-functionalized magnetic beads (SA-MB) through non-covalent π-π stacking interaction between 1-pyrenebutanoic acid (PBA) and Hyp. Briefly, the target DNA (tDNA) was hybridized to morpholino oligonucleotide (MO) that was immobilized on the surface of SA-MB. PBA was incorporated to the backbone of tDNA by means of phosphate-zirconium-carboxylate coordination reaction. Then, fluorescent Hyp was linked to PBA via π-π stacking interaction. Under the optimal conditions, this biosensor displayed a good linear relationship between the fluorescence intensity and the tDNA concentrations in the range from 1 to 100 nM with a low detection limit of 2.9 nM. The mismatch type in a specific DNA sequence can also be efficiently discriminated. Compared with some other reports, this method is more convenient, and has great potential for practical applications.