Among low viral load (DNA of hepatitis B virus (HBV) was <2000IU/mL), the factor of the loss of hepatitis B surface antigen (HBsAg) remained elusive. The retrospective study recruited patients with chronic hepatitis B (CHB) who were negative low for hepatitis B e-antigen (HBeAg), had a low viral load, and experienced HBsAg loss during follow-up. CHB patients with low-viral load but without consequent HBsAg loss were also enrolled at the ratio of 1:4. The factors contributing to HBsAg loss were analyzed. A total of 80 patients were recruited for the current study, with a mean age of 63.9years and 61.3% being male. Among them, 62.5% patients (50/80) were treated with potent nucleoside/nucleotide analogues (NAs) during the follow-up period. Additionally, 12.5% patients (10/80) had a prior history of NAs treatment before enrolment. During the follow-up, HBsAg loss occurred in 17 patients (21.3%). Compared with patients without HBsAg loss, those with HBsAg loss were younger (57.9years vs 65.5years; P=0.01), had lower HBV DNA levels (1.3 log10 IU/mL vs 2.3 log10 IU/mL; P=0.003), and higher proportion of prior NAs-treated history. Logistic regression analysis revealed that the factors associated with factors associated with HBsAg loss were age <60years (OR/CI: 3.95/1.15-13.60, P=0.03), prior NAs-treated history (OR/CI: 7.59/1.42-40.51, P=0.01) and current NAs-treated (OR/CI: 0.19/0.05-0.71, P=0.01). In the study, older age and prior NAs were positively associated with HBsAg loss, and current NAs was negatively associated with HBsAg loss. Additionally, some patients experienced HBsAg loss during the NAs therapy.