Crude Amaryllidaceae alkaloids (AAs) extracted from Lycoris radiata are reported to exhibit significant anti-cancer activity. However, the specific alkaloids responsible for the pharmacodynamic activity and their targets still remain elusive. In this context, we strived to combine affinity ultrafiltration with topoisomerase I (Top I) as a target enzyme aiming to fish out specific bioactive AAs from Lycoris radiata. 11 AAs from Lycoris radiata were thus screened out, among which hippeastrine (peak 5) with the highest Enrichment factor (EF) against Top I exhibited good dose-dependent inhibition with IC50 at 7.25 ± 0.20 μg/mL comparable to camptothecin (positive control) at 6.72 ± 0.23 μg/mL. The molecular docking simulation further indicated the inhibitory mechanism between Top I and hippeastrine. The in vitro antiproliferation assays finally revealed that hippeastrine strongly inhibited the proliferation of HT-29 and Hep G2 cells in an intuitive dose-dependent manner with the IC50 values at 3.98 ± 0.29 μg/mL and 11.85 ± 0.20 μg/mL, respectively, and also induced significant cellular morphological changes, which further validated our screening method and the potent antineoplastic effects. Collectively, these results suggested that hippeastrine could be a very promising anticancer candidate for the therapy of cancer in the near future.