Background: Head and neck squamous cell carcinoma (HNSCC) is often diagnosed at an advanced stage and has a dismal prognosis. Anti-PD1/PD-L1 checkpoint inhibitors have shown survival benefit for the treatment on HNSCC, and are better tolerated than chemotherapy. Unfortunately, the response rate of immunotherapy is low for advanced HNSCC. Therefore, the identification of predictive biomarkers of response to anti-PD1/PD-L1 is a key point for better selecting patients that would benefit from immunotherapy. Methods: The present study included 44 patients with advanced HNSCC who received anti-PD-1 immunotherapy. Follow-up was available for them. Primary tumor tissues and matched blood samples were collected. Genome profiles were analyzed using a designed 543-gene panel based on NGS, and were available on 39 of 44 patients. Profiles of infiltrating immune cells were available for 44 patients. The infiltrating intensity of PD-1, PD-L1, CD8, CD68 and CD57 positive cells were assessed by multiplex immunohistochemistry. Results: The most frequently mutated genes were TP53 (79%), CDKN2A (33%), NOTCH1 (21%), CASP8 (13%) and PIK3CA (10%). Patients with mutations in the PI3K pathway showed a trend of longer OS (P = 0.06). Moreover, the TMB-high group showed significant superior OS than the TMB-low group (p = 0.0018). PD-L1 expression was positively correlated with CD8 expression within stroma, and PD-1 expression correlated significantly with PD-L1 and CD8 expression within both tumor and stroma areas. In addition, tumor/stromal PD-L1 and PD-1 expression were both associated with better OS (P < 0.05). Prolonged OS was also observed in patients with more tumor and stromal infiltration of CD8⁺ cells, CD8⁺PD-L1⁺ cells, CD68⁺ macrophages, or nature killer (NK, CD57⁺) cells. Conclusions: Higher TMB, PD-L1 expression, as well as the infiltration of immune cells within both tumor and stromal area might serve as favorable prognostic markers in advanced HNSCC patients treated with anti-PD-1 immunotherapy.