In this paper, a novel phenyleneethynylene derivative 4,4′-(2,5-dimethoxy-1,4-phenylene)bis(ethyne-2,1-diyl) dibenzoic acid (p-acid) was synthesized. Infrared spectroscopy (IR), nuclear magnetic resonance (NMR), photoluminescence (PL) spectroscopy and ultraviolet visible (UV-vis) spectroscopy were applied to characterize p-acid. The p-acid doped silica nanoparticle (p-acid@SiO₂) was assembled in layer-by-layer (LBL) self-assembling processes using tetraethoxysilane (TEOS). Scanning electron microscope (SEM) and transmission electron microscope (TEM) were applied to characterize p-acid@SiO₂. To research the practical applicability, a sample DNA sensor was constructed using p-acid@SiO₂ label, in which the PL intensity response was proportion to the target-DNA (S₂) concentration in the range of 0.05–1000 fM, with a detection limit of 20 pM. Furthermore, the DNA sensor showed high specificity, excellent stability, and good reproducibility. The p-acid@SiO₂-based DNA sensor can also provide potential application for detection of other pathogen DNA.