This study aimed to evaluate the efficacy of Peg-interferon (Peg-IFN)-nucleoside analog (NA) sequential optimization therapy (SOT) in HBeAg-positive patients with chronic hepatitis B (CHB). In this prospective two-center study, 132 CHB patients were assigned to receive Peg-IFN standard therapy for 48weeks (65 patients) or Peg-IFN monotherapy for 12-24weeks and NA add-on for those without early virological response (EVR) (67 patients). Both patient groups were monitored and followed for 24weeks after treatments stop. At week 24 after treatments stop, the Peg-IFN-NA SOT group achieved more HBsAg levels drop (- 1.35 vs - 0.67 log10 IU/mL, p = 0.016), higher HBsAg ≤ 100IU/mL (32.8% vs 9.2%, p = 0.001), HBV DNA undetectable (79.1% vs 49.2%, p < 0.001), and ALT normalization (80.6% vs 38.5%, p < 0.001) rates compared with Peg-IFN monotherapy. At week 24 after treatments stop, nosignificantdifference was found in HBeAg seroconversion (35.8% vs 27.7%, p = 0.316), HBsAg loss (8.9% vs 4.6%, p = 0.323) and HBsAg seroconversion rates (4.5% vs 1.5%, p = 0.325) between Peg-IFN monotherapy group and Peg-IFN-NA SOT group. Starting with Peg-IFN followed by addition of NA achieved more HBsAg levels drop, and higher HBsAg ≤ 100IU/mL, HBV DNA undetectable, and ALT normalization rates compared with Peg-IFN monotherapy.