This study investigates preoperative serum cytokine levels in patients with oral squamous cell carcinoma (OSCC). The study included 51 patients with OSCC and 42 healthy controls (HCs). Serum samples of 12 cytokines were analyzed using a multiplex bead-based flow cytometry immunoassay. Mann-Whitney U test and binary logistic regression analysis were performed to identify significant indicators of OSCC. Receiver operating characteristic (ROC) curves evaluated the diagnostic performance.Spearman analysis was assessed the correlation between cytokines and tumor-node-metastasis staging of OSCC. Serum levels of interleukin (IL)-2, IL-5, IL-6, IL-8, IL-12P70, IL-17, and interferon gamma (IFN-γ) were significantly higher (P < 0.05) in patients with OSCC than in HCs. IL-5, IL-6, IL-8, IL-12P70, IL-17, IFN-γ, combination 1 (IL-6 and IL-8), and combination 2 (IL-6 and IL-12P70) had area under the curve (AUC) values > 0.7, with combination 2 exhibited the highest AUC of 0.995. Serum cytokine profiles were significantly different (P < 0.05) between the patients with OSCC and HCs. IL-5, IL-6, IL-8, IL-12P70, IL-17, IFN-γ, combination 1, and combination 2 effectively distinguished between HCs and patients with OSCC. Cytokine combinations enhanced OSCC diagnostic accuracy, with significantly elevated IL-6 levels (P < 0.05) in advanced-stage compared to early-stage OSCC, indicating its potential impact on disease progression and prognosis.