Updated project metadata. Patients with oral cavity squamous cell carcinoma (OSCC) are frequently first diagnosed at an advanced stage, leading to poor prognosis and high mortality rate. Early detection of OSCC using body fluid-accessible biomarkers may help improve the prognosis and survival rate of OSCC patients. As tumor interstitial fluid (TIF) is a proximal fluid enriched with cancer-related proteins, it is therefore a valuable reservoir suitable for the discovery of cancer biomarkers as well as dysregulated biological pathways in tumor microenvironment. Thus, we harvested and analyzed the paired tumor (TIF) and adjacent noncancerous (NIF) interstitial fluids from ten OSCC patients using one-dimensional gel electrophoresis coupled with the nano-liquid chromatography-tandem mass spectrometry (GeLC-MS/MS) approach and label-free spectral counting method. The results showed that 113 proteins were up-regulated in at least six OSCC TIFs compared to their respective NIFs, and that the aminoacyl tRNA biosynthesis was statistically enriched in TIFs by Gene Set Enrichment Analysis (GSEA). The enrichment of aminoacyl tRNA biosynthesis in OSCC was verified that four members (IARS, KARS, WARS, and YARS) were revealed to be elevated in OSCC by IHC (n = 12). Among the 113 up-regulated proteins, two candidates (NID1 and SERPINH1) were selected based on an integrated bioinformatics analysis and verified that the salivary NID1 levels were significantly higher in OSCC patients (n = 48) compared to health (n = 51) and OPMD individuals (n = 53). Moreover, the IHC results (n = 222) showed that the expression of NID1 was higher in OSCC compared to adjacent noncancerous epithelium and correlated with pathological T, N, and overall stage as well as survivals of OSCC patients. These results collectively indicate that the analysis of TIF is help to understand the tumor microenvironment, and that salivary NID1 is a potentially useful biomarker for the OSCC.