Surface-enhanced Raman scattering (SERS) has been intensely researched for many years as a potential technique for highly sensitive detection. This work, through the reduction of HAuCl(4) with pyrrole in aqueous solutions, investigated a facile one-pot synthesis of flower-like Au nanoparticles with rough surfaces. The formation process of the Au nanoflowers (AuNFs) was carefully studied, and a spontaneous assembly mechanism was proposed based on the time-course experimental results. The key synthesis strategy was to use pyrrole as a weak particle stabilizing and reducing agent to confine crystal growth in the limited ligand protection region. The nanometer-scale surface roughness of AuNFs provided several hot spots on a single particle, which significantly increased SERS enhancement. Good biocompatible stable Raman-active probes were synthesized by coating AuNFs with chitosan. The conservation of the SERS effects in living cells suggested that the chitosan-capped AuNFs could be suitable for highly sensitive detection and have potential for targeting of tumors in vivo.