To evaluate the probable predicting factors and biomarkers associated with the relapse of chronic hepatitis B after withdrawal of nucleos(t)ide analogs, a clinical assessment of patients with chronic hepatitis B, either hepatitis B virus e antigen (HBeAg)-positive or -negative, who had received the treatment of nucleos(t)ide analogs and met the requirement for withdrawal was made. Then the clinical data were collected including sex, age, treatment course, HBeAg level, baseline alanine aminotransferase (ALT) and HBV DNA levels, liver stillness, HBV surface antigen (HBsAg) level, interleukin 21 (IL-21) level and frequency of peripheral blood CD4^＋ IL-21^＋ T cells at the time of withdrawal. Forty seven patients met the requirement for withdrawal. Among them, 26 patients were HBeAg-positive and 21 patients were HBeAg-negative. The mean age was (42.45±14.08) years. The mean treatment course was (4.60±1.61) years. The mean baseline ALT level was (174.38±200.84) U/L. The mean baseline HBV DNA level was (5.87±1.06) log copies/mL. The mean HBsAg level was (1 104.82±1 328.11) IU/mL. The mean IL-21 level was (729.82±427.39) pg/mL. The mean frequency of peripheral blood CD4^＋ IL-21^＋ T cells was (0.79±0.49)%. The liver stillness measurement at the time of withdrawal was F1 in 27 patients, F2 in 16 patients, and F3 in 4 patients. Twenty seven (57.45%) patients relapsed in one year. Single-factor logistic regression analysis showed that the relapse of chronic hepatitis B after withdrawal of nucleos(t)ide analogs was associated with treatment course, IL-21 level, frequency of peripheral blood CD4^＋ IL-21^＋ T cells, and HBsAg level. Multi-factor logistic regression analysis showed that HBsAg level and IL-21 level could predict the relapse of chronic hepatitis B after withdrawal of nucleos(t)ide analogs.