A simple, ultrasensitive, highly selective, and reagent-free aptamer-based biosensor has been developed for quantitative detection of adenosine triphosphate (ATP) using surface-enhanced Raman scattering (SERS). The sensor contains a SERS probe made of gold nanostar@Raman label@SiO(2) core-shell nanoparticles in which the Raman label (malachite green isothiocyanate, MGITC) molecules are sandwiched between a gold nanostar core and a thin silica shell. Such a SERS probe brings enhanced signal and low background fluorescence, shows good water-solubility and stability, and exhibits no sign of photobleaching. The aptamer labeled with the SERS probe is designed to hybridize with the cDNA on a gold film to form a rigid duplex DNA. In the presence of ATP, the interaction between ATP and the aptamer results in the dissociation of the duplex DNA structure and thereby removal of the SERS probe from the gold film, reducing the Raman signal. The response of the SERS biosensor varies linearly with the logarithmic ATP concentration up to 2.0 nM with a limit of detection of 12.4 pM. Our work has provided an effective method for detection of small molecules with SERS.