For thousands of years, corn silk has been widely used as an antidiabetic, antioxidant, and antihyperlipidemic and for other effects, but there is a lack of studies that correlate the extracts of flavonoid composition with their biological activities. Thus, the objectives of this study were to optimize the conditions for extracting flavonoids, identify flavonoids, and correlate the flavonoid composition with the biological activities in corn silk. The response surface experiments showed that the highest flavonoid content was predicted at 45.321 min, 57.349°C, 26.089 mL/g, and 71.269%, respectively. The verification experiment results under these optimized conditions showed an ultrasonic time of 45 min, an ultrasonic temperature of 57°C, a liquid-to-material ratio of 26, and an ethanol volume fraction of 70%. No significant differences (the relative error is 4.378%) were observed between the theoretical and experimental TFC values, indicating that the developed models were accurate. Under these optimum extraction conditions, 20 major compounds were identified and quantified by UPLC-LTQ/Orbitrap MS. Furthermore, these optimum ethanol extracts of corn silk are effective against and hypoglycemic activity compared with the traditional heating reflux extraction method. Six corn silk components seem to be the main contributors to the inhibitory effect against and hyperglycemia activities. These results are useful for the application of corn silk in the food or pharmaceutical industry.