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A facile photoreduction strategy to attach silver nanoparticles on filter paper (AgNPs@FP) to form surface-enhanced Raman scattering (SERS) substrate array was proposed and examined. As prepared, SERS substrate array was applied to detect low concentration of adenine and other nucleobases in aqueous solutions. In addition, the formed SERS substrate array allows mass analysis of aqueous samples with a requirement of only 10 μL in sample volume. To optimize and understand the parameters in preparations, factors such as the concentrations of citrate and silver nitrate, photoreduction time, and concentration of sodium hydroxide were varied and examined. Para-nitrothiophenol was used as a Raman probe molecule and scanning electron microscope (SEM) images of the substrates were used to explore the influences of experimental factors in the preparation of SERS array. Results indicated that silver ions could be effectively reduced and deposited in/on filter paper with the presence of citrate. The formed AgNPs@FP exhibits a three-dimensional structure as the particles formed on and beneath the surface of the cellulosic fibers could be observed. Deposition of analyte on restricted substrate array rendered reproducible results and the spot-to-spot SERS intensity varied within 8%. At optimal conditions, the substrate enhancement factor approached 107. We demonstrated that the filter paper-based SERS substrates can be used as a suitable tool for biological analysis, and a limit of detection better than 160 nM was obtained in detection of adenine molecules in aqueous solutions with a linear range up to 20 μM. Copyright © 2014 John Wiley & Sons, Ltd.