Background: Patients with advanced biliary tract cancer (BTC) have a poor prognosis, and high unmet medical needs still exist for the treatment of this disease. Nivolumab has shown its effectiveness against multiple cancer types, especially those with PD-L1 expression. This study assessed the efficacy and safety of nivolumab in BTC patients with a combined positive score (CPS) ≥ 1 who were refractory or intolerant to the standard of care (SOC): gemcitabine, cisplatin and tegafur/gimeracil/oteracil (S-1). Methods: This was a single-arm, multicenter, open-label, prospective phase II study. Key eligibility criteria were as follows: having unresectable or recurrent BTC (intrahepatic bile duct cancer, extrahepatic bile duct cancer, gallbladder cancer, or ampullary cancer); being refractory or intolerant to SOC; having adequate hepatic, renal and hematological function; and having a CPS ≥ 1 in central assessment. Patients received nivolumab (480 mg, every 4 weeks) until disease progression, clinical deterioration, or unacceptable toxicity. The primary endpoint was objective response rate (ORR) assessed by the central review according to RECIST 1.1. The secondary endpoints included overall survival (OS), progression-free survival (PFS), and safety. Results: A total of 84 patients were enrolled between Mar 11, 2020 and Mar 15, 2021, of them 12 received one regimen and 72 received two or more regimens. The median follow-up period was 6.90 months. ORR per central assessment was 10.7% (CR: 3.6%, PR: 7.1%), and ORR per investigator assessment was 15.5% (CR: 2.4%, PR: 13.1%). The median PFS per central and investigator assessment was 1.02 (95% CI 0.95–1.77) months and 2.48 (95% CI 1.87–2.83) months, respectively. The median OS was 6.90 (95% CI 5.26–9.20) months. The median duration of response per central and investigator assessment was not reached (range 2.8–13.0+) and 10.15 (range 2.0–12.8+) months, respectively. The 1-year survival rate was 25.3% (95% CI 15.4–36.4). The most frequent treatment-related adverse events were pyrexia (19.0%), pruritus (16.7%), and decreased appetite (13.1%). Conclusions: Nivolumab may have some activity with a durable response and a manageable safety profile in heavily treated BTC patients with CPS ≥ 1. Clinical trial information: JapicCTI-205097.