Background A relevant study found that allergic rhinitis (AR) may be related to the imbalance of nasal flora. Therefore, if the nasal flora of AR patients can be detected quickly, it is of great significance to study the distribution law of nasal flora in AR patients and explore its correlation with AR. Objective To design a new and convenient nano-DNA sensor for quick screening of nasal flora in allergic rhinitis (AR) patients, so as to provide experimental basis for the prevention and treatment of AR. Methods We create a synthesized nanostructured DNA biosensor called Nano-TiO2-DNA sensor which can be combined with samples from nasal mucosa or secretion with high efficiency and detect certain flora in situ without DNA extraction or RNA sequencing. In a physical property test, firstly, we tested the permeability, solubility, and storage temperature of nano-TiO2, so as to provide experimental basis for the synthesis of Nano-TiO2-DNA sensor. Subsequently, the permeability of Nano-TiO2-DNA sensor in Staphylococcus aureus was further tested. In a clinical experiment, we selected 60 AR patients treated in our hospital from September 2020 to September 2021 as the AR group and 60 healthy people who underwent physical examination at the same time as the control group. The Nano-TiO2-DNA sensor was used to detect typical nasal flora in AR patients, and Pearson's correlation analysis was used to explore the correlation between nasal flora with serum IgE and eosinophils. Results As for physicochemical characteristics, this sensor can permeate into certain bacteria directly and specifically. It has high affinity ability with a target, and the combination can be detected by evaluating the released fluorescence qualitatively and quantitatively. It can be stored at −20°C in ethyl alcohol stably. By this sensor, we have successfully detected Staphylococcus aureus, Klebsiella pneumoniae, and viridans streptococci in AR patients compared with healthy people, which will help these patients in the prevention of acute sinusitis and acute or subacute pneumonia. Furthermore, we found Proteus had the strongest positive correlation with AR while Actinomyces had the biggest negative correlation. Conclusion The Nano-TiO2-DNA sensor will help an outpatient doctor more for quick screening certain nasal flora in AR patients and improve the prevention of AR-related complications.