AbstractIn this research, by simultaneously regulating the two major factors affecting the plasmonic enhanced fluorescence (PEF), spectral overlap and the distance between the fluororophores and the noble metal nanoparticles, a significantly enhanced fluorescent signal is achieved. Core‐shell nanostructures composed of aspect ratio (AR) adjustable gold nanorods (GNRs) and various thickness of SiO2 are prepared and the decorated fluorophores are realized optimized PEF. A typical stimuli‐responsive conjugated polymer, polydiacetylene (PDA), and a near‐infrared (NIR) dye Cy5.5 are selected as fluorophores and their fluorescent signal are enhanced 7.26 and 4.41 times, respectively. Based on the optimized optical properties, a multifunctional antibody modified Mab‐Cy5.5‐GNRs@SiO2 is successfully demonstrated the targeting, imaging, and photothermal therapy (PTT) effects on SKOV‐3 ovarian cancer cells.