ABSTRACTStrawberry (Fragaria × ananassa) is a popular fruit with rich nutrients and a delicious taste. But this fruit is very vulnerable to diseases and decay. Therefore, L‐phenylalanine (Phe) (0, 4, 8 mM) was considered to improve biochemical characteristics and activity of antioxidant enzymes in strawberry fruit cv. Sabrina under cold storage (5, 10, 15 days). After treatment and storage, traits including weight loss, total phenol (TP), antioxidant capacity, ascorbic acid, total anthocyanin (TA), total flavonoid (TF), malondialdehyde (MDA), soluble protein content and antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase (POD) and phenylalanine ammonialyase (PAL) were evaluated at 5‐day intervals. Our findings showed that the treatment of L‐phenylalanine in different concentrations prevented the weight loss of the fruit compared to the control and maintained and increased TP, antioxidant capacity, ascorbic acid, TA, TF, soluble protein and SOD, CAT, APX, POD, and PAL enzymes activity. Also, Phe decreased the MDA content and peroxidation of lipid. The results showed that 4 mM Phe is the best treatment for improving phytochemical characteristics and maintaining fruit quality. The findings indicated that Phe treatment may be useful to improve quality and increase postharvest shelf life in strawberry fruits.