In this work, we propose a new method for the large-scale production of flexible, periodic alumina arrays with well-ordered pores. We show the incorporation of pre-patterning based on polystyrene (PS) nanosphere lithography into an aluminium anodization process. We prepared ordered monolayers of PS spheres with average diameters of (510 ± 10) nm and (430 ± 10) nm on a large area (1.5 × 1.5 cm2) of the Si substrate. Next, we deposited a 5 μm aluminium layer on arrays of PS nanospheres using the sputtering technique. After the deposition, we covered the aluminium film with a polymer Scotch adhesive tape, and separated it from the silicon substrate by ultrasonic-assisted lift-off. Finally, we performed the anodization of the aluminium. We compared the pore and cell sizes, and the pore distance for the templates obtained by this technique, with reference to the templates prepared by a two-step anodization process. Using this new approach, we obtained highly ordered hexagonal 2D lattices over a large area of up to 2 cm2 with sparse defects, amounting to not more than four defects per 1000 μm2 on average. Here, we show that the use of indentation techniques is not necessary and can be replaced by a fast, cheap and easy pre-patterning step based on nanosphere lithography.