Silicon nanowire (SiNW) field-effect transistor (FET) biosensors have already been used as powerful sensors for the direct detection of disease-related biomarkers. However, the multiplexed detection of biomarkers in real samples is still challenging. Interleukin 8 (IL-8) and tumor necrosis factor α (TNF-α) are two typical biomarkers of oral squamous cell carcinoma (OSCC). In this study, we developed a multiplexed detection methodology for IL-8 and TNF-α detection in saliva using SiNW FET biosensors. We fabricated the SiNW FET sensors using a top-down lithography fabrication technique. Subsequently, we achieved the multiplexed detection of two biomarkers in saliva by specific recognition of the two biomarkers with their corresponding antibodies, which were modified on the SiNW. The established method was found to have a limit of detection as low as 10 fg/mL in 1 × PBS as well as 100 fg/mL in artificial saliva. Because of its advantages, including label-free and multiplexed detection, non-invasive analysis, highly sensitive and specific determination, the proposed method is expected to be widely used for the early diagnosis of OSCC.