Surface enhanced Raman Spectroscopy (SERS), as an analytical method, has the notable advantages of high sensitivity, high selectivity, high reproducibility and non-destructivity. It has been widely used in component characterization, environmental science, biomedicine, biosensors and other fields in the recent decades. Among them, gold and silver and other noble metal nanoparticles-based films have been widely used for SERS active substrates. A key factor in SERS is how to design and fabricate SERS substrates with large size, high enhancement, high repeatability, and recyclability. In general, the combined effect of the electromagnetic enhancement of the noble nanoparticles in a regular array and the enhancement of the electromagnetic coupling among the nanoparticles enhance the detection performance of the SERS substrate. However, the surface roughness of the nanostructures prepared by conventional methods such as photolithography and electron beam lithography is not enough. In this paper, we developed a method for the fabrication of gold nanoparticles tetragonal array pore structure by the combination of photolithography and chemical replacement methods. And, the electromagnetic enhancement properties SERS signal of the fabricated Au nanoparticles array substrates were investigated. The regular round holes array were etched with a tetragonal lattice structure on the silicon by photolithography. Then, the Fe film was deposited on the holes array by magnetron sputtering. After that, the chloroauric acid liquid membrane with a concentration of 1.893 8 mol·L-1 was spin-coated into the holes array on the substrate. The Fe film was then replaced with Au nanoparticles array in the iron holes array. Thus, the fabrication of SERS substrate with gold nanoparticles square lattice was completed. Based on the scanning electron microscopy (SEM) of the microstructure of the substrate, the gold nanoparticles deposited on the substrate were more and larger with the increasing reaction time between the iron film and the chloroauric acid solution. In order to verify the difference in nanostructure caused by different replacement time, the electromagnetic enhancement properties of the prepared gold nanoparticles array was characterized with SERS measurements. The SERS spectra of Au nanostructured substrate were measured by using rhodamine 6G (R6G) as probe molecule. The experimental results showed that the gold nanoparticles were more compact and order with a longer replacement time, and we can get the corresponding stronger SERS signal.