Surface-assisted laser desorption/ionization mass spectrometry (SALDI MS) has been successfully applied in the analysis of various small molecules. In this work, gold nanoparticles/thiol-β-cyclodextrin-functionalized TiO nanowires (AuNPs/SH-β-CD-TiO NWs) were prepared to enhance the performance of SALDI MS and mass spectrometry imaging (MSI). A monolithic TiO film was first grown on an indium tin oxide (ITO) glass slide via a modified sol-gel method and treated in an alkaline environment to form nanowires. TiO NWs were chemically modified by SH-β-CD for immobilizing AuNPs densely and strongly. Compared with the conventional organic matrix 2,5-dihydroxybenzoic acid (DHB), the prepared AuNPs/SH-β-CD-TiO NWs showed superior performances on detection sensitivity, repeatability, and analyte coverage. Analytes typically detectable with negative-ion matrix-assisted laser desorption/ionization (MALDI) MS could also be observed using AuNPs/SH-β-CD-TiO NWs in the positive ion mode. Its successful usage efficiently enhanced the SALDI MS detection of various small molecules such as carbohydrates, fatty acids, and bile acids in the positive ion mode. The developed SALDI substrate was further used to characterize and discriminate the natural and in vitro cultured Calculus Bovis, as well as natural and artificial Moschus. Furthermore, the spatial distribution of several natural products in spearmint leaves and potato tubers was explored by tissue imprinting and deposition on the AuNPs/SH-β-CD-TiO NW surface for SALDI MSI in dual-polarity mode, respectively. The wide application and satisfied detection sensitivity make AuNPs/SH-β-CD-TiO NWs ideal for SALDI MS and MSI of various natural products.