Gastric cancer is one of the most common malignant digestive cancers, and its diagnostic has still faced challenges based on metabolic analysis due to complex sample pretreatment and low metabolite abundance. In this study, inspired by the structure of bovine omasum, we in situ synthesized a novel interfacial carbon-based nanocomposite of graphene supported nickel nanoparticles-encapsulated in the nitrogen-doped carbon nanotube (Ni/N-CNT/rGO), which was served as a novel matrix with enhanced ionization efficiency for the matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) saliva metabolic analysis of gastric cancer. Benefiting from its high sp2 graphitic degree, large surface area, strong UV absorption, and rich active sites, Ni/N-CNT/rGO matrix exhibited excellent performances of reproducibility, coverage, salt-tolerance, sensitivity, and adsorption ability in MALDI-TOF MS. The differential scanning calorimetry (DSC) and thermal conversion behaviors explained the highly efficient LDI mechanism. Based on saliva metabolic fingerprints, Ni/N-CNT/rGO assisted LDI MS with cross-validation analysis could successfully distinguish gastric cancer patients from healthy controls through the screening of four potential biomarkers with an accuracy of 92.50%, specificity of 88.03%, and sensitivity of 97.12%. This work provided a fast and sensitive MS sensing platform for the metabolomics characterization of gastric cancer and might have potential value for precision medicine in the future.