Background: Combination strategy of immune checkpoint inhibitors has potential to replace chemotherapy as a new first-line therapy for advanced biliary tract cancer (BTC). The aims of this study were to assess the efficacy of anti-PD-1 combination therapy in the first-line treatment of advanced BTC and to find reliable biomarkers associated with overall survival (OS). Methods: Clinical data from patients with advanced BTC who received chemotherapy or anti-PD-1 combination therapy as first-line were collected. The primary outcome of this study was OS. Blood test results were collected from patients who received anti-PD-1 in the week prior to treatment to calculate inflammation scores and analyze their relationship with OS. A total of eight scores were selected for analysis, including platelet-to-lymphocyte ratio (PLR), neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-C-reactive protein (CRP) ratio (LCR), lymphocyte-to-monocyte ratio (LMR), CRP to-albumin ratio (CAR), systemic immune-inflammation index (SII), prognostic nutritional index (PNI), and pan-immune-inflammation value (PIV). Moreover, archival tissues were collected for genetic testing and immunohistochemical staining to analyze genetic changes and the infiltration of immune cells in tumor tissues in relation to survival. Results: From January, 2018, to July, 2022, 64 patients were recruited: 11 were in the chemotherapy group, 21 were in the anti-PD-1 plus chemotherapy group, 21 were in the anti-PD-1 plus targeted therapy group and 11 were in the triple group (anti-PD-1 plus chemotherapy and targeted therapy). The median overall survival (mOS) was 7.9 months, 11.3 months, 12.8 months and 28.7 months, respectively. Compared to first-line chemotherapy, mOS was significantly prolonged in the triple group (p= 0.031). The results of inflammation scores showed that patients with high LCR (≥0.4), high LMR (≥2.5), low CAR ( < 0.18), high PNI (≥44.3) or low PIV ( < 480.9) had significantly prolonged mOS compared to those with different scores (p< 0.05). Genetic testing results suggested that patients with poor survival all had TP53 mutations and had higher levels of KRAS and ERBB2 mutations. Immunohistochemical results showed that a low FOXP3/CD8 ratio (≤0.27) was associated with prolonged OS (p= 0.029). With CD4-low, CD8-high, CD56-positive, CD163-high, FOXP3-high and MPO-high in TME as one factor, we calculated immunoscore according to the number of factors. The high immunoscore group ( > 2) showed significantly superior OS (p= 0.003). Conclusions: First-line anti-PD-1 antibody combination therapy was superior to chemotherapy, and triple therapy significantly improved survival. Peripheral blood immune-inflammation score, FOXP3/CD8 ratio and immunoscore have potential as biomarkers for predicting the efficacy of first-line anti-PD-1 antibody therapy in advanced BTC.