The lack of a cultivation system for human noroviruses (HuNoV) is a major barrier to understand virus biology and the development of effective antiviral strategies. The study aims to verify the reported replication of the reverse genetics system of HuNoV G Ⅱ. 3 U201, and further to seek a better sequence from the HuNoV clinical strains to construct a highly efficient infectious clone. First the sequences of HuNoV GⅡ.3 U201 genome with T7 promoter and EF­1α promoter were synthesized, and inserted into the vector, named pU201 and pEF­1α­U201, respectively. Meanwhile, negative control plasmids with viral RNA polymerase activity inactivated were constructed. To verify whether these clones could replicate, T7 polymerase was co­transfected with pU201 and pEF­1α­U201 was transfected alone in COS 7 cells and Huh 7 cells, respectively, and HuNoV RNA level was determined by real­time quantitative polymerase chain reaction (RT­qPCR) in different time post transfection. The results showed that the HuNoV RNA levels of pU201 and pEF­1α­U201 were nearly two times and three times higher than those of the negative control group respectively. To facilitate the detection of HuNoV replication, full­length infectious clones with NanoLuc™ luciferase (Nluc) reporter gene were constructed by inserting Nluc sequence in front of viral genome, named pU201­Nluc and pEF­1α­U201­Nluc. COS 7 cells and Huh 7 cells were transfected and Nluc activity was determined in different time post transfection. The results showed that the Nluc activity of pEF­1α­U201­Nluc was nearly 2 times higher than those of the negative control group and the inhibitor­treated group, but there was no significant increase in pU201­Nluc. These results suggested that EF­1α promoter was superior to T7 promoter in initiating HuNoV GⅡ.3 U201 replication. Finally, to seek a better HuNoV sequence, a full­length infectious clone was constructed with a clinical isolate of HuNoV GⅡ.4 genome from a Taiwan patient and EF­1α promoter, named PCTc. COS 7 cells and Huh 7 cells were transfected and HuNoV RNA level was determined in supernatant and cells by RT­qPCR. And the viral RNA level between PCTc and control groups had no significant difference. The results suggested that full­length infectious clones and subgenomic clone of HuNoV GⅡ.3 U201 could only achieve limited and unsustainable replication in cells, and replication capacity may also be associated with different sequences of virus strains.