Au nanoporous films (NPFs) with different surface modification and morphology were fabricated and utilized as substrates for the analysis of a series of compounds, including amino acids, drug, cyclodextrins, peptides, and polyethylene glycols, using surface-assisted laser desorption/ionization time-of-flight mass spectrometry (SALDI-TOF MS). It was found that the size and interconnection state of the NPF ligament as well as the surface modification are key parameters that affect the laser desorption/ionization performance. Compared with 2,5-dihydroxybenzoic acid, pristine NPF, and aminobenzenethiol or 3-mercaptopropanoic acid modified Au NPFs, cysteine modified Au NPF generated intense and background-suppressing mass spectra. Regarding the effect of Au NPF morphology, the Au NPF with nanopores in the range of 10-30 nm, ligament size of 5 nm, and electrochemistry surface area of 26.1 m(2)/g exhibited the highest performance as a substrate. This high-performance NPFs can be easily fabricated by capping agent replacement induced self-organization of ultrathin nanowires, followed by self-assembling of a monolayer (SAM) of cysteine. The good thermal/electroconductivity and uniformity of Au NPFs avoided the fragmentation of analytes, eliminated the intrinsic matrix ions interference, and provided good reproducibility (RSD ≤ 10%). Additionally, the fabricated NPFs can be easy divided into microarrays (a ~4 × 4 array from a 1 cm × 1 cm NPF). This work provides a simple and cost-effective route for acquiring an Au nanostructure as a SALDI substrate, which offers a new technique for high-speed analysis of low-molecular weight compounds.