A new flexible, super-hydrophobic, and highly sensitive polydimethylsiloxane (PDMS) based-substrate would be developed for surface-enhanced Raman spectroscopy (SERS) detection. PDMS substrate is modified by nitrogen plasma and then deposited the organic conjugated molecules (α, ω-diperfluorohexylquaterthiophene, DFH-4T) films various thickness of Au nanoislands. The perfluorohexyl (-n-C₆F₁₃) chain of DFH-4T displays the advantages of high volatility and low vapor deposition temperature and can form a super-hydrophobic ivy-like nanostructure. After depositing the Au nanoislands on DFH-4T, the vertically arranged Au-DFH-4T nanostructure combined provides a tip-focusing effect and generates a very high electromagnetic field to enhance the Raman signal. Benefiting from the hydrophobic ivy-like nanostructure (maximum contact angle: ~146^o) of Au@DFH-4T nanostructure, the hydrophobic molecule (vitamin D₃, cholecalciferol) can be measured at ultralow concentration (10⁻¹⁰ M). Moreover, the flexible SERS sensor exhibited acceptable efficiency and durability after 100 cycles bending tests. SERS intensity would increase ~1.6 time at 0.35 cm bending radius. This facile and efficient detection technique for meeting the increased demands for bio-detection, especially for hydrophobic biomolecules.