Background: To evaluate anlotinib combined with toripalimab for advanced biliary cancer that failed or without the will of standard first-line treatments. Methods: Open single arm phase II study were conducted, eligible patients (pts) were advanced biliary without the chance of operation and failed or without the will of standard first-line treatments. Pts received anlotinib 12mg po qd in 21-day cycles and toripalimab 240mg i.v. every 3 weeks. The main outcome measure was the objective response rate (ORR) of the target lesions evaluated by researchers based on the RECIST 1.1 standard, the secondary outcome measures were progress free survival (PFS) and treatment-related adverse event (TRAE). Results: As of Jan, 2022, 15 pts were enrolled including 9 the intrahepatic cholangiocarcinoma, 3 hilar Cholangiocarcinoma, and 3 the gall bladder carcinoma. 11 (73.3%) pts failed standard first-line treatments, and 4 pts were without the will of standard first-line treatments. The median follow-up was 254 days and 15 pts were all evaluated. 4PR, 9SD and 2PD were achieved. The ORR and DCR for irradiated target lesions was 26.7% and 86.7%, respectively. PFS for first line treatment was 139 days, and for second line treatment as 171 days. By this strategy, 1 ps was successfully transferred to operation after 3 cycle treatment. TRAEs (all grades) occurred in 14 (93.3%) pts. Grade 3 related AEs were fatigue (13.3%). Grade 1-2 related AEs were white blood cell count decreased (53.3%), hypertension (20.0%), thrombocytopenia (60.0%), fatigue (13.3%), hypothyroidism (6.7%), and fever asthenia (6.7%). Conclusions: The combination of anlotinib and toripalimab were demonstrated promising anti-tumor activity in advanced biliary cancer. Clinical trial information: ChiCTR2000037847.