A surface-enhanced Raman scattering (SERS) substrate composed of silver nanoparticles (Ag NPs) was synthesized by spark discharge deposition (SDD). The spark-ablated species were directly deposited on a copper substrate at room temperature and atmospheric pressure. Sharp and reproducible SERRS signals from the model molecule Rhodamine B (Rh B) were observed from the prepared substrates. Under optimized deposition conditions, the detection limit of Rh B reached the single molecular level (1.0 × 10−16 M) with an enhancement factor of 3.9 × 1012. This substrate also provided excellent SERS activity toward crystal violet molecule and the representative biomolecule adenine. We were further interested to find that the activity of the prepared substrate responded very sensitively when we changed the morphology of the deposited NPs by adjusting the operating parameters. An increase in the spark time to 20 min increased the particle size and filling factor, which induced the particles to grow into self-assembled dendrite-like structures. We observed the highest intensity in an Ag NP film with an average particle size of 45 nm and filling factor of 0.57. We speculate that the high sensitivity of this substrate may have derived from the high electromagnetic field produced by the tuning of the particle size and the formation of nanogaps (hot spots) between adjacent particles. This method offers a simple, cost effective, eco-friendly route to the synthesis of highly sensitive, reproducible SERS substrates.