Objective:　To　study　the　inhibitory　acitvity　of　phosphorothioate　antisense　oligonucleotide　(P-ASODN)　prop5　against　influenza　virus　entry　into　cells.　Methods:　A549　cells　infected　by　influenza　virus　were　used　in　this　study.　A　random　sequence　(prop5R)　and　a　sense　sequence　(prop5S)　served　as　negative　controls.　Culture　media　supernatants　were　collected　to　detect　copies　of　viral　RNA　by　real-time　quantitative　RT-PCR.　Further,　the　effect　of　prop5　on　virus　attachment　was　validated　by　hemagglutination　inhibition　assay　and　its　effect　on　virus　entry　was　determined　by　hemolysis　inhibition　assay.　Rusults　Prop5　could　inhibit　infection　of　A549　by　influenza　A/Jingfang/1/86(H1N1).　Prop5　did　not　interfere　with　influenza　virus　attachment　on　cells.　Prop5　could　inhibit　membrane　fusion　mediated　by　influenza　A/Jingfang/1/86(H1N1),　A/Lufang/9/93(H3N2),　A/FM/1/47(H1N1)　and　A/PR/8/34(H1N1).　Conclusion:　Prop5　blocks　influenza　virus　entry　in　sequence-independent　manner.　This　extra　activity　enhances　the　anti-influenza　virus　capability　of　prop5.