Laser interference lithography (LIL) was used to fabricate gold nanodisk arrays on glass which was studied as surface-enhanced Raman spectroscopy (SERS) substrates. The Raman response of varying array periods (250 nm, 300 nm, 344 nm, 395 nm, and 446 nm) was compared using the chemical rhodamine 6G. The 300-nm period displayed the highest SERS enhancement among the tested LIL substrates. Experimental transmission measurements and their finite element method (FEM) simulations were taken to understand the optical response of the LIL substrates. The 300-nm-period LIL substrate was used to detect urea to confirm its practical use as a SERS substrate. The enhancement factor of the 300-nm-period substrate was 2.3 x 106. Furthermore, the detection limit of urea was 0.05 mM for the optimized substrate.