Serum hepatitis B virus (HBV) RNA is a novel biomarker for evaluating treatment response. Detailed information regarding serum HBV RNA kinetics during treatment with nucleos(t)ide analogues (NAs) is limited.

To ascertain serum HBV RNA kinetics during long‐term NA treatment and identify associated factors.

We enrolled 76 HBeAg‐positive chronic hepatitis B patients receiving NA from randomised controlled trials. Laboratory assays were undertaken every 3 months. Factors associated with serum HBV RNA kinetics were identified by generalised estimating equations.

Baseline serum HBV RNA was 8.5 ± 1.0 log₁₀ copies/mL. Decline in serum HBV RNA during NA therapy was biphasic: the first phase (HBV DNA detectable) had a fast decrease (median slope, −0.207 log₁₀ copies/mL/month) and was followed by a second phase (HBV DNA undetectable) with slow decrease (median slope, −0.071 log₁₀ copies/mL/month). In the first phase, factors independently associated with lower initial serum HBV RNA were male sex (OR, 0.685, P = 0.044), low baseline HBsAg (OR, 0.525, P = 0.001) and rapid virological response (RVR) (OR, 0.624, P = 0.031). In the second phase, only RVR was independently associated with serum HBV RNA kinetics, including its lower initial level (OR, 0.694, P = 0.043) and greater decline (OR, 0.966, P = 0.002). Based on viral dynamics, time needed to achieve undetectable serum HBV RNA from baseline was 43.56 (IQR: 29.49‐66.40) months.

RVR was a significant determinant for biphasic decline in serum HBV RNA during NA treatment, which significantly influenced the treatment duration required to achieve undetectable serum HBV RNA.