Mass spectrometry imaging (MSI) has made the spatio-chemical characterization of a broad range of small-molecule metabolites within biological tissues possible. However, available matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) suffers from severe background interferences in low-mass ranges and inhomogeneous matrix deposition. Thus, surface-assisted LDI-MS (SALDI-MS) has been an attractive alternative for high-sensitivity detection and imaging of small biomolecules. In this study, we construct a new composite substrate, hydrophobic polydopamine (hPDA)-modified TiO₂ nanotube (TDNT) coated with plasmonic gold nanoparticle (AuNP-hPDA-TDNT), as a dual-polarity SALDI substrate using an easy and cost-effective fabrication approach. Benefitting from the synergistic effects of TDNT semiconductor and plasmonic PDA modification, this SALDI substrate exhibits superior performance for dual-polarity detection of a vast diversity of small molecules. Highly reduced background interferences, lower detection limits, and spot-to-spot repeatability can be achieved using AuNP-hPDA-TDNT substrates. Due to its unique imprinting performance, various metabolites and lipids can be visualized within jatropha integerrima petals, ginkgo leaves, strawberry fruits, and latent fingerprints. More valuably, the universality of this matrix-free substrate is demonstrated for mapping spatial distribution of lipids within mouse brain tissue sections. Considered together, this AuNP-hPDA-TDNT material is expected to be a promising SALDI substrate in various fields, especially in nanomaterial development and life sciences.