Background: Both Pharmacopoeia of the People's Republic of China and the ancient Chinese herbal formulas recorded that mulberry leaves collected after frost had good quality. However, the reason has not yet been fully elucidated. Objective: We investigated the effect of frost on the accumulation of flavonoids and antioxidant activities of mulberry leaves. Materials and Methods: Liquid chromatography-mass spectrometry and high-performance liquid chromatography were used to analyze chemical components and determine the content of five flavonol glycosides from mulberry leaves collected before and after frost, respectively. Antioxidant activities of the same mulberry leaves were evaluated by total antioxidant capacity (TAC), Fe2+ equivalent (FeE), reducing power (RP), 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay, and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) free radical scavenging assay. Results: Ten compounds were identified as flavonol glycosides exception of chlorogenic acid. Quantitative analysis showed that content of isoquercitrin, astragalin, and kaempferol-3-O-(6''-acetyl)-β-D-glucopyranoside reached a maximum of 3.05 mg/g, 0.70 mg/g, and 0.69 mg/g after Frost's Descent, respectively. Moreover, the lowest value of flavonol glycosides appeared in August. The antioxidant activities were also found to have the same tendency. The maximum value of TAC, FeE, RP, DPPH and ABTS were 64.3 rutin equivalent (RE) mg/g, 46.2 RE mg/g, 31.3 RE mg/g, 22.5 RE mg/g and 26.7 RE mg/g, respectively, in November. They were 1.4 times, 1.4 times, 1.6 times, 1.6 times, and 1.9 times of the minimum values, respectively, in August. There was a significantly and positively correlation between antioxidant activities and content of flavonol glycosides (P < 0.01). Conclusion: Frost is beneficial to the accumulation of flavonol glycosides and the improvement of antioxidant activities of mulberry leaves.