Oral cavity squamous cell carcinoma (OSCC), which is a leading cause of cancer-related death worldwide, is frequently associated with poor prognosis and mortality. The discovery of body fluid-accessible biomarkers may help improve the detection of OSCC. In the present work, we established primary cell cultures derived from OSCC and adjacent noncancerous epithelium and performed comparative profiling of their secretomes. Using spectral counting-based label-free quantification, we found that 64 proteins were significantly higher in primary OSCC cells compared with primary adjacent noncancerous cells. We then retrieved the mRNA expression levels of these 64 proteins in oral cavity tumor and noncancerous tissues from public domain array-based transcriptome data sets and used this information to prioritize the biomarker candidates. We identified 19 candidates; among them, the protein levels of THBS2, UFD1L, and DNAJB11 were found to be elevated in OSCC tissues compared with adjacent noncancerous epithelia. Importantly, higher levels of THBS2 in OSCC tissues were associated with a higher overall pathological stage, positive perineural invasion, and a poorer prognosis. Moreover, the salivary levels of THBS2 in OSCC patients were elevated compared to those of noncancer controls. Our results collectively indicate that analysis of the primary cell secretome is a feasible strategy for biomarker identification, and that THBS2 is a potentially useful salivary marker for the detection of OSCC.