Lateral flow assays, as a low-cost, simple, portable and disposable product of vitro diagnostic, are being widely used for point-of-care testing. However, the poor sensitivity of LFAs is the main challenge for commercialization. In order to enhance the sensitivity of LFAs, cellulose nanofibers (CNFs) have been integrated into LFAs to enhance the sensitivity of protein LFAs. A simple method is alsopresented to modify the properties of paper substrate by incorporating CNFs into a nitrocellulose membrane to enhance thesensitivity of nucleic acid LFAs. This method changes the pore size, porosity, surface groups and surface area of paper substrate and then increases the adsorption ability of biomolecules on paper substrate. The results indicate that the sensitivity of nucleic acid LFAs in Staphylococcus aureus testing achieves a20-fold enhancement. Hence, we anticipate that this simple method has the potential for other paper-based devices to improve the performance. Graphical abstractA simple method is used to modify the properties of paper substrate by incorporating cellulose nanofibers (CNFs) into nitrocellulose (NC) membrane to enhance the sensitivity of nucleic acid LFAs.