Background and Purpose Gut microbes play an important role in the occurrence of lung cancer, immunotherapy, and chemotherapy. In this study, we analyzed the characteristics of gut microbes in patients with lung cancer and investigated the effect of gut microbes on anti-PD-1 therapy combined with chemotherapy. Methods Fecal samples from 21 non-small cell lung cancer (NSCLC) patients and 22 healthy volunteers who were treated in the Fourth Hospital of Hebei Medical University from 2019 to 2021 were collected. DNA was extracted from all samples, and the V3-V4 region of the bacterial 16S rRNA gene was PCR-amplified using the Illumina sequencing platform, and R language was used for data analysis. Results There were significant differences in the Beta diversity and metabolic pathways of gut microbes between NSCLC patients and healthy individuals (p < 0.05). Bifidobacterium, Escherichia, and Sarterella were significantly enriched in patients with clinical benefit response (p < 0.05), and these three bacteria had certain predictive value for clinical benefit. Patients with Bifidobacterium breve had significantly longer median progression-free survival (mPFS) compared with patients with no detectable Bifidobacterium breve feces at baseline (106 days vs. NR, p < 0.001). Multivariate COX analysis showed that the presence of B.breve was an independent good prognostic factor affecting the PFS of patients receiving combination therapy (p < 0.05). Conclusion The clinical efficacy of anti-PD-1 therapy combined with chemotherapy in Chinese advanced NSCLC patients is closely related to the gut microbiota, and Bifidobacterium breve may be a potential biomarker to predict the efficacy of immune-combined chemotherapy.