Infection with hepatitis B virus (HBV) is a major cause of liver diseases such as cirrhosis and hepatocellular carcinoma. In our previous studies, we identified indole derivatives that have anti-HBV activities. In this study, we optimize a series of 5-hydroxy-1H-indole-3-carboxylates, which exhibited potent anti-HBV activities, using three-dimensional quantitative structure-activity relationship (3D QSAR) studies with comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). The lowest energy conformation of compound 3, which exhibited the most potent anti-HBV activity, obtained from systematic search was used as the template for alignment. The best predictions were obtained with the CoMFA standard model (q2 = 0.689, r2 = 0.965, SEE = 0.082, F = 148.751) and with CoMSIA combined steric, electrostatic, hydrophobic and H-bond acceptor fields (q2 = 0.578, r2 = 0.973, SEE = 0.078, F = 100.342). Both models were validated by an external test set of six compounds giving satisfactory prediction. Based on the clues derived from CoMFA and CoMSIA models and their contour maps, another three compounds were designed and synthesized. Pharmacological assay demonstrated that the newly synthesized compounds possessed more potent anti-HBV activities than before (IC50: compound 35a is 3.1 μmol/l, compound 3 is 4.1 μmol/l). Combining the clues derived from the 3D QSAR studies and from further validation of the 3D QSAR models, the activities of the newly synthesized indole derivatives were well accounted for. Furthermore, this showed that the CoMFA and CoMSIA models proved to have good predictive ability.