In order to explore the key factors for the nutritional quality evaluation of raw milk in Shanghai, a total of 167 batches of raw milk were collected from 11 large-scale dairy farms in Shanghai from July 2022 to March 2023, and their amino acid composition, fatty acid composition, vitamins, lactoferrin and trace elements were detected. The amino acid composition data were analyzed by means of principal component analysis (PCA) and the proportions of various fatty acids were compared. The results showed that 16 amino acids, 26 fatty acids, active factors including lactoferrin, α-lactalbumin and β-lactoglobulin, and 9 essential trace elements were detected in raw milk. On this basis, we selected 13 key factors for the nutritional quality evaluation of raw milk, which could provide an important reference for the further screening and utilization of raw milk sources in Shanghai.