Abstract Objective This prospective clinical trial used improved microbead-assisted flow cytometry (iMAF) to detect plasma extracellular vesicles (EVs) PD-L1/PD-L2 expression, aiming to validate their static predictive and dynamic monitoring value for neoadjuvant immunochemotherapy efficacy in locally advanced head and neck squamous cell carcinoma (HNSCC). Methods Ninety-two locally advanced HNSCC patients were prospectively enrolled, with a 69-patient dynamic cohort. The iMAF assay was optimized for EVs PD-L1/PD-L2 detection. Correlations between biomarkers and pathological response, progression-free survival (PFS), and overall survival (OS) were statistically analyzed. Results The iMAF technology for EVs PD-L1/PD-L2 expression was successfully established and optimized with 20,000 beads and 80 μL plasma. High pretreatment EVs PD-L1 and EVs PD-L2 were independent predictors of favorable pathological response (all AUC>0.7, p<0.001), especially in CPS>1 subgroups. High EVs PD-L1/PD-L2 expression was significantly associated with better PFS and OS (2-year PFS 96.9% vs 84.7%/ 100% vs 65.1%,2-year OS 95.7% vs 85.4%/100% vs 77.2%, p<0.05). Decreased EVs PD-L2 during treatment predicted higher response rate and better survival (2-year PFS 100% vs 65.1%,2-year OS 100% vs 77.2%,p<0.05). Conclusion We demonstrate that pretreatment plasma EVs PD-L1/PD-L2 expression are promising static predictive biomarkers, and decreased EVs PD-L2 serves as a reliable dynamic indicator. The optimized iMAF technology enables convenient and precise immunotherapy prediction and dynamic monitoring for HNSCC. Key words: Head and neck squamous cell carcinoma; extracellular vesicles; microbead-assisted flow cytometry (iMAF); PD-L1/PD-L2; neoadjuvant immunochemotherapy; predictive value