A facile procedure was introduced to prepare uniform and highly active substrates assembled with gold nanoparticles for surface-enhanced Raman spectroscopy (SERS). The laser power and time dependent SERS study of 4-mercaptopyridine (4-MPy) adsorbed on this substrate was investigated. The relative intensity of the characteristic Raman peak pair (1575 and 1610 cm鈥?) was found to change with the laser power and the exposure time. The spectral changes may originate from change in the fraction of the double-end-bonded and the single-end-bonded states of 4-Mpy adsorbed on the gold nanoparticle-assembled substrate. In addition, this study offers a way to avoid the effect of laser power to achieve reliable spectral response to other factors.