Although the approved hepatitis B virus (HBV)‐polymerase inhibitors (e.g., lamivudine) often lead to drug‐resistance, several natural products have shown promising efficacies. Though Aloe vera (AV) gel and its constituents are shown inhibitors of many viruses, their anti‐HBV activity still remains elusive. We therefore, tested the anti‐HBV potential of AV extract and its anthraquinones in hepatoma cells, including molecular docking, high‐performance thin layer chromatography (HPTLC), and cytochrome P450 (CYP3A4) activation analyses. Our anti‐HBV assays (HBsAg/HBeAg Elisa) showed maximal inhibition of viral antigens production by aloe‐emodin (~83%) > chrysophanol (~62%) > aloin B (~61%) > AV extract (~37%) in HepG2.2.15 cells. Interestingly, the effect of aloe‐emodin was comparable with lamivudine (~86%). Moreover, sequential treatment with lamivudine (pulse) followed by aloe‐emodin (chase) enhanced the efficacy of monotherapy by ~12%. Docking (AutoDock Vina) of the anthraquinones indicated strong interactions with HBV‐polymerase residues that formed stable complexes with high Gibbs's free energy. Further, identification of aloe‐emodin and aloin B by validated HPTLC in AV extract strongly endorsed its anti‐HBV potential. In addition, our luciferase‐reporter gene assay of transfected HepG2 cells showed moderate induction of CYP3A4 by aloe‐emodin. In conclusion, this is the first report on anti‐HBV potential of AV–derived anthraquinones, possibly via HBV‐polymerase inhibition. Of these, although aloin B exhibits novel antiviral effect, aloe‐emodin appears as the most promising anti‐HBV natural drug with CYP3A4 activating property towards its enhanced therapeutic efficacy.