Finding reliable markers for predicting the efficacy of immunotherapy is urgently needed. We sought to investigate the association between serum microRNAs (miRNAs) and checkpoint inhibitor response in non-small cell lung cancer (NSCLC). Discovery assay with sera miRNA profiling was performed, demonstrating 27 sera miRNAs (relative fold > 2, p < .05), 22 higher expressed and 5 lower expressed miRNAs, were differentially expressed in 19 responders compared to those in 27 non-responders. Further validation validated miR-93, -138-5p, - 200, - 27a, - 424, - 34a, - 28, -106b, -193a-3p, and -181a were significantly higher expressed (p < .01) in an independent cohort of 17 responders vs. 17 non-responders. Longitudinally, responders had increased sera expression levels of miR-93, -138-5p, - 200, - 27a, - 424, - 34a, - 28, -106b, -193a-3p, and -181a from pre-treatment to post-treatment (p < .01). More importantly, statistically significant improvement in PFS of patients was associated with the 10-high expressed miRNA pattern (median PFS of 6.25 versus 3.21 months, p < .001; hazard ratio, HR, 0.45; 95% CI, 0.25-0.76). Further OS improvement was also significantly associated with the 10-high expressed miRNA pattern in responders versus non-responders (median OS of 7.65 versus 3.2 months, p < .001, HR, 0.39; 95% CI, 0.15-0.68). In conclusion, these results demonstrated that alterations in circulating miRNAs are associated with the response and outcome in NSCLC patients treated with anti-PD1 drugs.