Background: Recent advances in cancer genome analysis and the practice of precision medicine have made it possible to identify fractions with rare genetic alterations. EGFR-amplified cancers are known to be rare fractions across organs and have a poor prognosis. The use of anti-EGFR antibody for EGFR-amplified cancers has been promising, but the evidence is not yet clear. Case: In this report, we describe the case of a 48-year-old man diagnosed with advanced gallbladder cancer. The patient was administered Gemcitabine plus Cisplatin, followed by S-1 monotherapy; however, disease progression was observed after two cycles of each regimen. Comprehensive genomic profiling test revealed EGFR-amplification, and the patient was treated with combination therapy with the anti-EGFR antibody Necitumumab, Gemcitabine, and Cisplatin. After two cycles of treatment, showed a reduction in tumor size, and the treatment response was evaluated as partial response. On day 90, after five cycles of treatment, tumor progression was confirmed. In addition, after disease progression, liquid biopsy revealed acquired pathogenic gene alterations suggesting anti-EGFR antibody resistance. Conclusions: This report supports the clinical benefit of anti-EGFR antibody for EGFR-amplified biliary tract cancers and the importance of genomic analysis in personalized therapy and drug resistance research.