Surface-enhanced Raman scattering (SERS) spectroscopy is a sensitive sensing technique. It is desirable to have an easy method to produce SERS-active substrate with reproducible and robust signals. We propose a simple method to fabricate SERS-active substrates with high structural homogeneity and signal reproducibility using electron beam (E-beam) lithography without the problematic photoresist (PR) lift-off process. The substrate was fabricated by using E-beam to define nanograting patterns on the photoresist and subsequently coat a layer of gold thin film on top of it. Efficient and stable SERS signals were observed on the substrates. In order to investigate the enhancement mechanism, we compared the signals from this substrate with those with photoresist lifted-off, which are essentially discontinuous gold stripes. While both structures showed significant grating-period-dependent fluorescence enhancement, no SERS signal was observed on the photoresist lifted-off gratings. Only transverse magnetic (TM)-polarized excitation exhibited strong enhancement, which revealed its plasmonic attribution. The fluorescence enhancement showed distinct periodic dependence for the two structures, which is due to the different enhancement mechanism. We demonstrate using this substrate for specific protein binding detection. Similar periodicity dependence was observed. Detailed theoretical and experimental studies were performed to investigate the observed phenomena. We conclude that the excitation of surface plasmon polaritons on the continuous gold thin film is essential for the stable and efficient SERS effects.