Selective fabrication of metallic nanostructures at the spotting area is required to increase the signal-to-background noise ratio (SBR) of the metal-enhanced fluorescence (MEF) substrate. As a simple and cost-effective fabrication method for MEF substrate with high SBR, a glancing angle deposition (GLAD) process of Ag material on the UV-imprinted micropost array (50 μm in height, 300 μm in diameter, and 600 μm in pitch) was proposed to selectively fabricate Ag nanorods on the top of micropost structure (spotting area). Ag nanorod formation at the bottom of the micropost decreased as the deposition angle in Ag GLAD increased. A deposition angle of 89° and deposition thickness of 500 nm were selected as the optimum GLAD conditions to maximize the SBR. The optimum Ag nanorods on micropost array (AgNMPA) MEF substrate provided 71-fold fluorescence signal enhancement and 25-times higher SBR than the bare glass substrate. It also provided 7-times higher SBR than the Ag nanorod MEF substrate, which has a similar Ag nanorod structure but is not selectively formed. The detection limit of AgNMPA was 16- and 4-times lower than that of the amine-functionalized glass substrate and commercial epoxy slide, respectively. Although the fluorescence signal of AgNMPA was similar to that of Ag nanorod substrate, the detection limit was 2-times lower because of the low signal standard deviation caused by the low background noise and clear spot shape.