Background: Efficacy and prognosis of chemotherapy in first-line setting for patients with advanced biliary tract cancer (BTC) was dismal currently. Recently, PD-1/PD-L1 blockades combined with dual chemotherapy in first-line setting exhibited superior efficacy for patients with BTC. Nevertheless, it's important to highlight that both the TOPAZ-1 and KEYNOTE-966 trials had reported a median OS of less than 13 months. This underscored the necessity for further advancements to meet the demands of clinical practice. Anlotinib, a novel multitarget TKI primarily targeting VEGFR1-3, demonstrated promising therapeutic activity as second-line therapy for patients with BTC. Therefore, this study was designed to explore the efficacy and safety of anlotinib plus TQB2450 (PD-L1 blockade) combined with nab-paclitaxel and cisplatin as first-line therapy in advanced BTC. Methods: Patients with previously untreated metastatic or locally advanced BTC (ICC, ECC, GBC) were recruited and treated with anlotinib (10mg, po, d1~14, q3w) and TQB2450 (1200mg, iv, d1, q3w) plus nab-paclitaxel (200mg/m², iv, d1, q3w) and cisplatin (60mg/m², iv, d1, q3w) until disease progression or unacceptable toxicity. The tumor response was assessed according to RECIST 1.1 using CT scans every two cycles. Adverse events were recorded by severity in accordance with the NCI CTC AE Version 5.0. The predefined sample size was 20. Primary endpoint was ORR and secondary endpoints included safety, DCR, PFS, OS and biomarker explore. Results: From April 2023 to Aug 2023, a total of 18 patients were enrolled, 14 patients who had received first tumor response were included in this analysis. The best overall response indicated that there were 6 PR (42.9%), 7 SD (50.0%) and 1 PD (7.1%). Therefore, the preliminary ORR was 42.8% (95%CI: 17.7%-71.1%), DCR was 92.9% (95%CI: 66.1%-99.8%). Median PFS of the 14 patients was not yet reached. Additionally, safety profile exhibited that the regimen was tolerable. The most common treatment-emergent adverse events among the 18 patients with the incidence >30% were leukopenia (67%), fever (39%), pain in extremity (33%) and malaise (28%). Common grade ≥3 treatment-emergent adverse events were leukopenia (28%), fever (6%), malaise (6%), stomachache (6%), oral mucositis (6%), thrombocytopenia (6%), vomit (6%), rash (6%) and hypotension (6%). Conclusions: Preliminary results suggested that anlotinib plus TQB2450 combined with nab-paclitaxel and cisplatin as first-line therapy in advanced BTC exhibited encouraging efficacy and manageable adverse events. The conclusion should be validated in more patients consecutively. Clinical trial information: NCT05812430.