The persistent toxic substances (PTS), pollutants that have environmental persistence and toxic effects such as carcinogenicity and endocrine disruption, are generally detected by the chromatographic method. In this work, hydrophobic gold nanostructures were fabricated via simple electrochemical deposition and could be used for direct SERS detection of PTS with a portable Raman spectrometer. The obtained hydrophobic substrates free from modification show high affinity towards PTS, such as polycyclic aromatic hydrocarbons (PAHs), polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs). According to the Cassie equation, the wetting properties of the surface changed from hydrophilic to hydrophobic when the contact angle changed from 77° to 123°, increasing the deposition time. The SERS signals collected on sixteen randomly selected points show that the relative standard deviation of the SERS intensity is 9.0%, indicating the substrate had good uniformity. Quantitative SERS detection of PTS was achieved, as the log–log plot of SERS intensity to PTS concentration exhibited a good linear relationship in view of the Freundlich equation. Quantitative analysis of fluoranthene, BDE-15 and PCB-15 was accomplished in the concentration range of 0.02–200 μM, 0.02–200 μM, 0.04–440 μM, with the detection limits of 6.7 nM, 2.6 nM and 5.3 nM, respectively. The effective SERS substrates provide a rapid and sensitive platform for trace level detection of hydrophobic contaminants in the environment.