MicroRNAs (miRNAs) are stably present in human serum. The relationship between circulating miRNAs and hepatitis B virus (HBV) infected liver disease has not been previously reported. Applied Biosystems array-based miRNA expression profiling was performed on pooled sera obtained from identified groups of chronic asymptomatic carriers (ASC), patients with chronic hepatitis B (CHB) and HBV-associated acute-on-chronic liver failure (ACLF), as well as healthy controls (HC). Nine miRNAs were verified in more clinical samples by RT-PCR. The correlation between miRNAs expression and the relationship between miRNA levels and clinical characteristics was analysed. Results showed that circulating miRNAs were detected in all disease and control samples, and their numbers increased with symptom severity, from 37 in HC, 77 in ASC, 101 in CHB, to 135 in ACLF. The expression levels of most miRNAs were also up-regulated in HBV-infected patients when compared to HC. Expression of the liver-specific miR-122 was significantly up-regulated in HBV-infected patients. Concomitant regulation of miRNAs not in clusters was disrupted by HBV infection. However, such disruption was not observed for miRNAs in paralogous clusters. Furthermore, the level of miRNAs in the CHB serum was up-regulated most in hepatitis B e antigen-positive patients. The expression levels of miR-122 and miR-194 correlated negatively with the age of patients with CHB or ACLF. Functional analysis showed that miR-122 could inhibit HBV replication in Huh7 and HepG2 cells. In all, our study revealed that a number of miRNAs were differentially expressed during HBV infection and underscored the potential importance of miR-122 in the infection process.