Detection of target analytes with high sensitivity and reproducibility remains a challenge for surface-enhanced Raman scattering (SERS) due to the lack of cost-effective and highly sensitive substrates. In this study, a hydrophobic SERS substrate capable of concentrating nanoparticles and analytes was prepared by spin-coating lubricating liquid onto commercial paper. The condensation effect of the paper-based hydrophobic substrate induced aggregation of gold nanoparticles (Au NPs) to generate ‘‘hot spots’’ for SERS and to drive analytes to the hot-spot areas for more sensitive detection. The obtained SERS signal intensity was 5-fold higher than that obtained using common paper, and a detection limit (LOD) of 4.3 × 10−10 M for rhodamine 6G (R6G) was achieved. Randomly selected points on the substrate and different batches of substrates all exhibited high reproducibility, and the relative standard deviation (RSD) at 1362 cm−1 is approximately 11%. A further application of the hydrophobic substrate was demonstrated by the detection of cytochrome C within a linear detection range of 3.90 × 10−8 M–1.25 × 10−6 M. In addition, the prepared substrate can obtain identifiable SERS spectra of cancer cells and non-cancer cells because a large number of AuNP or Au NPs clusters can adhere to cells, resulting in the construction of a 3D hotspot matrix. The disposable hydrophobic paper substrate eliminates the problem of solution diffusion, and also provides an effective platform for biomolecular screening detection.