Abstract: Objective: To establish a quality control method of Malus prunifolia (Willd.) Borkh. from the Naxi ethnic group so as to provide a basis for further study. Methods: In accordance with the Technical Guidelines for Research on Quality Standards of Chinese Medicinal Materials in Yunnan Province, qualitative identification was carried out by means of macroscopic characteristics, microscopic identification and thin-layer chromatography (TLC); moisture, total ash and extract content were detected by the methods specified in the 2020 edition of Chinese Pharmacopoeia, and the contents of hyperoside and quercetin were determined by high-performance liquid chromatography (HPLC). Results: The characteristics of the Malus prunifolia (Willd.) Borkh. were notable. The main microscopic features included numerous stone cells, calcium oxalate crystals, outer fruit skin cells, reticulate structures, lenticular tracheids, and numerous starch granules. The preliminary physicochemical tests indicated that the fruit contains flavonoids, phenols, organic acids, lactones, coumarins, tannins, oils, glycosides, and sugars, and may also contain volatile oils, amino acids, and protein compounds, though further research is needed to confirm these findings. TLC results showed that Malus prunifolia (Willd.) Borkh. herbs from different origins exhibited identical color spots at corresponding positions, with good reproducibility. The water content and total ash content in Malus prunifolia (Willd.) Borkh. were set at no more than 18.0% and 3.7%, respectively, and the hot water extractable substances should not be less than 6.78%. A method for simultaneously detecting and quantifying hyperoside and quercetin in Malus prunifolia (Willd.) Borkh. was established. Hyperoside showed a good linear relationship within the range of 0.02 mg/mL to 1 mg/mL, with the regression equation Y=22327x+54.678, and R²=0.9991; quercetin showed a good linear relationship within the range of 0.02 mg/mL to 1 mg/mL, with the regression equation Y=34550x+78.251, and R²=0.9992. Based on the test results of 12 batches of samples, it was proposed that the content of hyperoside in Malus prunifolia (Willd.) Borkh., calculated on dry weight basis, should not be less than 0.013%, and the content of quercetin should not be less than 0.038%. Conclusion: Malus prunifolia (Willd.) Borkh. has significant identification characteristics in terms of plant morphology, medicinal properties, microscopic characteristics and physicochemical characteristics. A method for simultaneous detection of hyperoside and quercetin in Malus prunifolia (Willd.) Borkh. has been established. The obtained data provide a theoretical basis for the formulation of quality standards of this medicinal material and further research and development of this medicinal resource.