Oviductus Ranae is a nutritional product for both medicine and food. Its quality is uneven due to multiple factors. An efficient method was established to evaluate the quality of Oviductus Ranae using fingerprint techniques and chemometric methods based on the analysis of polyunsaturated fatty acids (PUFAs) in petroleum ether extract by high performance liquid chromatography (HPLC). During this process, 27 batches of Oviductus Ranae were analyzed by HPLC and two types of chromatographic fingerprints were established. The fingerprint that was obtained by matching six known peaks was used for the quantification of six PUFAs. Another fingerprint was obtained by matching sixteen peaks with a peak area ratio greater than 0.5% and it was used to classify the different qualities of Oviductus Ranae by further combining three different chemometric models. The 27 batches of Oviductus Ranae were divided into four categories, which was consistent with the analysis results of six PUFAs contents. The two high-quality samples with significantly higher contents were classified into one category, and samples with medium contents were divided into two categories, including eight and thirteen samples, respectively. The four inferior samples with lower contents were classified into one category. The results indicated that the newly developed method has potential application prospects for the quality evaluation of Oviductus Ranae.