The purpose of the current study is to evaluate the correlation and consistency of simultaneous amplification testing (SAT) and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for quantitative detecting serum hepatitis B virus RNA. 212 serum samples from Shanghai Public Health Clinical Center including 81 CHB patients with HBV DNA≥100 IU/mL, 76 CHB patients with HBV DNA 100 IU/mL and 55 patients without HBV infection were detected by SAT and qRT-PCR methods. In HBV DNA≥100 IU/mL CHB patients, 95.06% (77/81) samples were positive both in SAT and qRT-PCR method. SAT showed a relevantly good correlation and comparability with qRT-PCR (R2=0.803, CCC=0.882). Bland Altman analysis shows absolute bias was 0.1 log10 copies/mL and relative bias was 0.97%. The paired T test analysis of test results had no difference (t =1.617，P =0.110). In HBV DNA 100 IU/mL CHB patients, 98.68% (75/76) samples were positive in SAT method and 88.16% (67/76) samples were positive in qRT-PCR method. The statistical analysis of test results had difference (P<0.001). SAT showed a relevantly bad correlation and comparability with qRT-PCR (R2=0.326, CCC=0.438). Bland Altman analysis shows absolute bias was 0.5 log10 copies/mL and relative bias was 86%. The paired T test analysis of test results had difference (t =3.654，P<0.001). In 55 patients without HBV infection, all samples were negative in SAT method and qRT-PCR method. Correlation analysis shows strong correlation and consistency between SAT and qRT-PCR for CHB patients with HBV DNA≥100 IU/mL. Difference was found in the measurement of RNA using SAT and qRT-PCR for CHB patients with HBV RNA 100 IU/mL.