In order to screen and characterize aptamers against hepatitis C virus(HCV) NS3 helicase, an 81bp single stranded DNA (ssDNA) random library was subjected to 8 rounds of selection against HCV NS3 helicase by SELEX method. The selected aptamers were cloned and sequenced.The primary sequences of the aptamers were analyzed by Clustal W, and the affinities of aptamers to HCV NS3 helicase were determined. After 8 rounds selection, the percentage of the ssDNA pool bound to HCV NS3 helicase from 0.45% inceased to 29.5%. The primary sequences of the aptamers were divided into 5 families with 4 conserved sequences. The affinity of aptamer H2 to HCV NS3 helicase was the highest, with K_d values as low as 140 nmol/L. Aptamer H2 (10 μmol/L) inhibited approximately 44% of the helicase activity of HCV NS3 in vitro. The results suggest that aptamer against HCV NS3 helicase have been identified by means of SELEX methods from an 81bp single stranded DNA random library.