Well-ordered periodic nanostructures are excellent substrates for many surface-enhanced Raman spectroscopy (SERS) applications. Conventional fabrication approaches such as high precision electron beam lithography or focused ion beam produce high resolution nano-features with great reproducibility at the expense of low throughput. In this work, a highly sensitive and scalable AAO-nano-fibre (ANF) SERS substrate is demonstrated by optimising the second anodisation time of the standard two-step anodisation of aluminium and performing an additional wet etching step on the resulting AAO substrate. The optimised ANF substrate exhibits SERS sensitivity that surpasses the AAO nanoholes and the metal-film-on-nanoparticles substrates. A detection limit of 0.1 nM is achieved with a signal-to-noise ratio of 2.6-3 using a low excitation power of 0.1 mW. The ANF substrate exhibits an enhancement factor of 9.28 × 106 and a standard deviation of no more than 8%. The results indicate that the highly sensitive and scalable ANF substrate is a promising substrate for commercial SERS application.