In order to establish a fatty acid fingerprint of the seed oil from Akebia trifoliata and explore the differential characteristic indicators of fatty acid composition in the seed oil from different regions, a total of 18 batches of Akebia trifoliata seed oil from different regions were extracted using Soxhlet extraction method. The fatty acid composition and content were analyzed, and the Akebia trifoliata seed oil fatty acid fingerprint was established using the "Chinese medicine chromatographic fingerprint similarity evaluation system". Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed to analyze the differential characteristic indicators of Akebia trifoliata seed oil. The results indicated that, the main fatty acids in Akebia trifoliata seed oil were oleic acid, linoleic acid, and palmitic acid, with their relative contents exceeding 19%. The fatty acid fingerprint of Akebia trifoliata seed oil was established, the similarity between the samples and the standard fingerprint was above 0.994, indicating the high accuracy and reliability. Oleic acid, linoleic acid, and palmitic acid can be used as differential markers for preliminary discrimination of geographical origin. However, further analysis and determination should be done by considering more diverse characteristic results.