Based on the structural scaffolds of natural products, two series of flavonoid derivatives, for a total of twelve compounds, were designed and synthesized as potential human telomerase inhibitors. Using a modified TRAP-PCR assay, compound 5c exhibited the most potent inhibitory activity against human telomerase with an IC₅₀ value of less than 50 μM. In vitro, the results demonstrated that compound 5c had potent anticancer activity against five classes of tumor cell lines. The molecular docking and molecular dynamics analyses binding to the human telomerase holoenzyme were performed to elucidate the binding mode of active compound 5c. This finding helps the rational design of more potent telomerase inhibitors based on the structural scaffolds of natural products.