Abstract A two-probe tandem DNA hybridization assay including capture DNA 1, probe DNA 2, and target DNA 3 was prepared. The long-lived luminescent europium complex doped nanoparticles (NPs) were used as the biomarker. The complex included in the particle was Eu(TTA) 3(5-NH 2-phen)-IgG (ETN-IgG), the europium complex Eu(TTA) 3(5-NH 2-phen) linking an IgG molecule. Silica NPs containing ETN-IgG were prepared by the reverse microemulsion method, and were easy to label oligonucleotide for time-resolved fluorescence assays. The luminophores were well-protected from the environmental interference when they were doped inside the silica network. The sequences of Staphylococcus aureus and Escherichia coli genes were designed using software Primer Premier 5.0. Amino-modified capture DNA 1 was covalently immobilized on the common glass slides surface. The detection was done by monitoring the fluorescence intensity from the glass surface after the hybridization reaction with the NPs labeled probe DNA 2 and complementary target DNA 3. The sensing system presented short hybridization time, satisfactory stability, sensitivity, and selectivity. This approach was successfully employed for preliminary application in the detection of pure cultured E. coli, it might be an effective tool for pathogen DNA monitoring.