A surface-enhanced Raman scattering (SERS) active substrate for the detection of polycyclic aromatic hydrocarbons (PAHs) was developed, which used 25, 27-dimercaptoacetic acid-26, 28-dihydroxy-4-terbutyl calix[4]arene (DMCX) to functionalize a gold colloid film. This SERS-active substrate prepared by self-assembly method exhibits a high sensitivity, especially for the detection of PAHs. With the use of this SERS-active substrate and with the application of the shifted excitation Raman difference spectroscopy (SERDS) technique, Raman signals of pyrene and anthracene in aqueous solutions at low concentration level (500 pM) can be obtained. Moreover, because PAHs are blocked from being directly adsorbed on gold colloid by DMCX and the photochemical reactions of adsorbates are avoided, the Raman bands of PAHs adsorbed on DMCX-fuctionalized gold colloid film can be one-to-one correspondence with those of solid PAHs, and additionally, this SERS-active substrate can be easily cleaned and reused. The obtained results demonstrate that the DMCX-functionalized gold colloid films prepared by self-assembly method have great potential to be developed to an in situ PAHs detection substrate.