Background <p>Tumor heterogeneity and invasive sampling limit traditional tissue-based PD-L1 testing in advanced lung cancer. We explored a non-invasive alternative by assessing PD-L1 expression on circulating tumor cells (CTC PD-L1) via liquid biopsy to predict immunotherapy outcomes.</p> Methods <p>Fifty-two advanced lung cancer patients were recruited, and 38 individuals received a combination of chemotherapy and PD-1/PD-L1 inhibitor therapy, with serial blood samples (baseline, during treatment, and at progression) analyzed using the LiquidBiopsy^™ platform and CTC PD-L1 assay. Primary endpoints included objective response rate (ORR), progression-free survival (PFS), and overall survival (OS).</p> Results <p>CTC PD-L1 was identified on special phenotype with a ratio of CK/CD45 &gt; 1.7 and PD-L1/CD45 &gt; 3.2. At baseline, 71.2% (37/52) of patients had detectable CTCs, with 50.0% (26/52) being CTC PD-L1⁺. CTC PD-L1⁺ patients exhibited significantly higher ORR (84.2% vs. 36.8%), longer median PFS (16&#xa0;months vs 4&#xa0;months; HR = 0.28, 95% CI 0.096–0.78, <i>p</i> = 0.0041), and superior OS (median survival, undefined; HR = 0.19, 95% CI 0.05–0.74, <i>p</i> = 0.017). Multivariate analysis confirmed CTC PD-L1⁺ as an independent predictor of response (<i>p</i> = 0.003) and potential as a complementary biomarker to tPD-L1.</p> Conclusions <p>We developed a novel procedure to detect and characterize PD-L1 expression on CTCs, which was a feasible, non-invasive biomarker for potentially predicting the combination of chemotherapy and immunotherapy efficacy in advanced lung cancer, addressing tissue sampling limitations and enhancing patient stratification and monitoring.</p> <p><i>Trial registration</i>: The study was approved by the Medical Ethics Committee of Fourth Affiliated Hospital of Soochow University, with ethics approval number 220154 on 23th January, 2022 and retrospectively registered under clinical trial number ChiCTR2500096312 on 21th January, 2025.</p>