Nasopharyngeal carcinoma (NPC) is usually diagnosed at advanced clinical stages, resulting in poor outcomes. To discover serum biomarkers for improved NPC diagnosis and/or management, we simultaneously analyzed the NPC cell secretome and tissue transcriptome to identify candidate genes/proteins that are highly upregulated in NPC tissues and also secreted/released from NPC cells. Among the 30 candidates identified, 11 proteins were chosen for further validation using the serum samples from NPC patients and healthy controls, including cystatin A, cathepsin B, manganese superoxide dismutase and matrix metalloproteinase 2. The results showed that serum levels of all the four proteins were indeed higher in NPC patients versus healthy controls and that the use of a three-marker panel (cystatin A, manganese superoxide dismutase and matrix metalloproteinase 2) can contribute to a better NPC detection than each marker alone. In addition, a higher pretreated serum level of cystatin A was found to be associated with a higher nodal stage and poorer prognosis of NPC patients and cystatin A could modulate the migration and invasion of NPC cells in vitro. Altogether, our results indicate that analysis of both the cancer cell secretome and tissue transcriptome is a feasible strategy for efficient identification of novel NPC serum marker panel.