Ag nanoparticles with 2-dimensional (2D) array structure were fabricated via nanosphere lithography. The influence of Cr interlayer position and metal deposition direction on the array structure was systematically studied. It was found that the structure of the 2D Ag nanoparticle array with Cr interlayer was better than that without interlayers. When the Cr interlayer was deposited on the PS mask, the tips of the acquired triangle nanoparticles were much sharper, and the area of the nanoparticle array was much larger than the other cases. Moreover, the achieved nanoparticle array has a better uniformity and compactness in structure, and higher binding ability to the substrate when Cr and Ag deposition direction was perpendicular to the surface of the substrate. Further absorption spectrum experiment proved the improvement of the structure and feature of the 2D Ag nanoparticle array. All these are very crucial for the future modification and fabrication of biochemical sensors with the 2D Ag nanoparticle array.