Seroclearance of hepatitis B surface antigen (HBsAg) has been widely studied; however, seroconversion of HBsAg and characteristics of viral load among hepatitis B e antigen (HBeAg)-negative chronic infection patients after HBsAg lost is not clear. We performed a large-scale study in a HBeAg-negative chronic infection cohort to evaluate spontaneous HBsAg seroclearance incidence from October 2012 to April 2017 in Jiangsu province, China. We also elucidated the characteristics of HBsAg seroconversion and hepatitis B virus (HBV) DNA detectability among patients who cleared HBsAg. A total of 2997 HBeAg-negative chronic infection patients (mean age 52.3 ± 12.9 years at baseline) were included. With 10 519 person-years of follow-up, 348 patients successfully spontaneously cleared HBsAg, with an incidence rate of 3.31 per 100 person-years. Patients with HBV DNA detectable ~1999 IU/mL at baseline had a lower probability of HBsAg seroclearance relative to those with undetectable HBV DNA, with a hazard ratio of 0.31 (95% CI = 0.23, 0.41). HBsAg seroconversion occurred in 37.3% of those patients who cleared HBsAg. The geometric mean of anti-HBs among those with HBsAg conversion was 79.4 mIU/mL. Female had a higher HBsAg seroconversion rate (P = 0.011). Among those with HBsAg seroclearance, 11.2% still had HBV DNA levels of higher than 100 IU/mL. Patients with higher HBV DNA at baseline had a higher risk of detectable HBV DNA levels even after HBsAg seroclearance (P < 0.001). This study reveals HBsAg seroconversion rates and HBV DNA undetectability epidemiological characteristics of patients with HBsAg seroclearance and suggests that monitoring HBV DNA is needed when managing HBeAg-negative chronic patients, even after clearing HBsAg.