Generally, nano-scale patterned sapphire substrate (NPSS) has better performance than micro-scale patterned sapphire substrate (MPSS) in improving the light extraction efficiency of LEDs. Laser interference lithography (LIL) is one of the powerful fabrication methods for periodic nanostructures without photo-masks for different designs. However, Lloyd’s mirror LIL system has the disadvantage that fabricated patterns are inevitably distorted, especially for large-area twodimensional (2D) periodic nanostructures. Herein, we introduce two-beam LIL system to fabricate consistent large-area NPSS. Quantitative analysis and characterization indicate that the high uniformity of the photoresist arrays is achieved. Through the combination of dry etching and wet etching techniques, the well-defined NPSS with period of 460 nm were prepared on the whole sapphire substrate. The deviation is 4.34% for the bottom width of the triangle truncated pyramid arrays on the whole 2-inch sapphire substrate, which is suitable for the application in industrial production of NPSS.