A new radioactive positron emission tomography (PET) tracer uses inhibitors of fibroblast activation protein (FAPI) to visualize FAP-expressing cancer associated fibroblasts. Significant FAPI-uptake has recently been demonstrated in biliary tract cancer (BTC) patients. Target volume delineation for radiation therapy still relies on often less precise conventional magnetic resonance imaging (MRI) registered with computed tomography (CT) imaging, especially in locally advanced BTC patients. The need for improvement in precise tumor detection and delineation led us to innovatively use the novel FAPI-PET/CT for radiation treatment planning. Gross tumor volumes (GTVs) of five locally advanced BTC cases were contoured under FAPI-PET/CT method. MRI/CT was used to delineate tumors additionally. The differentiation in target definition was analyzed between FAPI-PET/CT-based GTVs and the manually MRI/CT-based GTVs. Target definition differed significantly between different imaging methods with mean dice similarity coefficients of 0.5527, mean Jaccard similarity coefficients of 0.4296 and mean volumetric overlap difference of 0.5704, while the mean volumes and standard deviations of GTVs were 18.12±15.10 cm3 and 38.44±24.72 cm3, based on FAPI-PET/CT and MRI/CT respectively (P = 0.102). There was a discordance and difference between the volumes of FAPI-GTVs-based GTVs and the manually contoured GTVs based on MRI/CT. Due to its high tumor to background contrast, FAPI-PET/CT seems to be a superior imaging modality compared to the current gold standard MRI/CT in BTC. For the first time, we demonstrate how FAPI-PET/CT could facilitate target definition and increases accuracy in radiation oncology in BTC. Limited to the sample size, we still need more large-scale data to support this view.