Hantaan virus (HTNV) is a major zoonotic pathogen that causes hemorrhagic fever with renal syndrome (HFRS) in Asia, especially in China. Shaanxi province, which is located in northwest of China, is one of the areas in China most severely afflicted with HFRS epidemics annually. This study aims to establish a quantitative RT-PCR (qRT-PCR) assay to detect HTNV both in cell culture and clinical serum samples. We established a SYBR Green I-based one-step qRT-PCR assay that targets the S segment of the HTNV genome for rapid detection and quantification. The HTNV cRNA standards were constructed by in vitro transcription, and the copy numbers of the HTNV cRNA were quantified. Standard curve was generated by determining the mean cycle threshold (Ct) values versus 10-fold serial dilutions of the HTNV cRNA over a range of 1 × 10(8) to 1 × 10(3) copies/μl. The standard curve had a reaction efficiency of 102.1%, a correlation coefficient (R(2)) of 0.998, and a slope of -3.273. The coefficient of variation (CV) of the intra- and inter-assays ranged from 0.68% to 3.00% and from 0.86% to 3.21%, respectively. The cycle intervals of the qRT-PCR assay between each dilution ranged from 2.9 to 3.8 cycles, and the lowest detection limit of the qRT-PCR assay was 10 copies/μl. The assay exhibited high specificity that was confirmed by melting curve analysis, and no cross reaction with the Seoul virus (SEOV) and other viruses (HBV, HCV and HIV) was observed. HTNV RNA was also detected in the 27 serum samples of clinical HFRS patients using the assay, and the HTNV RNA viral load ranged from 2.06 × 10(1) to 1.95 × 10(5) copies/μl. The SYBR Green I-based one-step qRT-PCR assay is a sensitive, specific, reproducible, and simple method for detecting and quantifying HTNV in cell culture and clinical samples.