So far only a few approaches have been reported for the successful synthesis of 3D flower-like hierarchical gold nanostructures. In this paper, a facile, seedless and one-pot approach to synthesize a new class of flower-shaped gold nanoparticles with sheet-like petals has been proposed by reduction of HAuCl4 with ascorbic acid in (1-hexadecyl)trimethylammonium chloride solution, in the presence of silver nitrate (AgNO3). The growth of petals can be controlled and tuned by adjusting the amount of silver nitrate in the growth solution, and the reaction progress and evolution of the petals were monitored by time-course measurements via UV-vis-NIR spectroscopy and SEM to observe the growth mechanisms of nanoparticles. Besides, their optical properties, especially SERS performances, were also studied. These SERS-active gold nanoparticles were further utilized in the design of bright SERS tags for selectively in vitro imaging cancer cells. The results indicate that these Au NFs can be used as good SERS substrates or to design sensitive SERS sensors.