We demonstrated the antioxidant activity, inhibitory activity against α-glucosidase, α-amylase, and the content of phenolic and flavonoid compounds from fresh and boiled bamboo shoot extracts. This research also first investigates the content of 2,4-DTBP from bamboo shoots and its kinetic mechanism against α-glucosidase and α-amylase through in vitro and in silico studies. Total phenolic and flavonoid content and antioxidant activity of fresh samples were higher than that of boiled samples. Inhibitory activity of bamboo shoot extracts against α-glucosidase and α-amylase was related to the content of 2,4-DTBP by the highest potent obtained from the fresh samples. 2,4-DTBP displays mixed-type inhibition and is located at + 1 to + 2 subsites for α-glucosidase and −1 to −3 subsites for α-amylase. Our findings indicate that bamboo shoots had the source of bioactive compounds related to human health and the 2,4-DTBP in bamboo shoots is a candidate compound for anti-diabetes-related enzymes.