We focused on the development of a hand-held pathogen-detection device using smartphone-embedded electronic elements combined with functionalized magnetic particles (MPs) and sepharose. To perform affinity chromatography for evaluating DNA amplicons, avidin-conjugated MPs and succinimide-linked sepharose were used with biotin-primers. To mimic the centrifugal-based affinity ligand chromatography, a smartphone-mountable low-power fan was plugged into the charging port of a smartphone. The charging port stably emitted electric current at 3.0 V, and the fan blades were modified for use as a portable rotor. Based on the binding variation of MPs with DNA amplicons, the position of MPs in sepharose changed significantly during centrifugation. The change in distance was optically analyzed using the illumination sensor of the smartphone with respect to the altered transmittance due to the MPs. Amplified genes from Escherichia. coli O157:H7 samples ranging from 1.0 × 101 to 1.0 × 106 colony-forming units could be rapidly and immediately detected by the naked eye using a simple smartphone-based optical device. The results indicated that this novel biosensing technique is suitable for use as a point-of-care testing device in both industrial and clinical fields.