Objective To establish a screening system of efficient small interfering RNA (siRNA) target sites directed against truncated region of human cytomegalovirus (HCMV) UL54 gene (UL54S) with siRNA expression vectors. Methods Two small hairpin RNA (shRNA) expression vectors targeting truncated region of HCMV UL54 gene were constructed based on pAVU6 + 27 vector, and cotransfected into AD293 cells with the fusion protein expression vectors pUL54S-enhanced green fluorescent protein (EGFP). The levels of mRNA and EGFP were evaluated by means of reverse transcription-polymerase chain reaction (RT-PCR), fluorescence microscopy and flow cytometry so as to assess the inhibitory efficiency of siRNA. Analysis of variance was applied to analyze the variance of total fluorescence intensity to screen out the efficient target sites of siRNA.Results shRNA expression vector psiUL54-2 and fusion protein expression vector pUL54S-EGFP were cotransfected into AD293 cells. The EGFP expression level in pUL54S-EGFP/psiUL54-2 cotransfected group was lower than that in pUL54S-EGFP/pAVU6 +27 cotransfected group after 48 h of transfection. Gel analysis showed that the mRNA relative level of UL54S was 19.6 after 48 h of psiUL54-2/pUL54S-EGFP cotransfection, which was significantly lower than those in pUL54S-EGFP/psiUL54-1 group (96.6) and control group (100.0). Cotransfection of psiUL54-1/pUL54S-EGFP for 48h didn't show any effects on the expression of fusion protein UL54S-EGFP (P>0. 05).While psiUL54-1/pUL54S-EGFP cotransfection inhibited the expression of fusion protein UL54S-EGFP(19.43×104±2.29×104vs27.89×104±5.50×104, P<0.01).Conclusion Thescreening system of efficient siRNA targeting truncated region of HCMV UL54S is established successfully. The 1532th-1550th nucleotide acids of UL54S coding sequence are efficient siRNA target sites.