The hepatitis B virus (HBV) particle is an icosahedral nucleocapsid surrounded by a lipid envelope containing viral surface proteins. A small domain (matrix domain, MD) in the large surface protein L and a narrow region (matrix binding domain, MBD) including isoleucine 126 on the capsid surface have been mapped where point mutations like core-I126A specifically blocked nucleocapsid envelopment. Possibly, both domains interact with each other during virion morphogenesis. By the SELEX method we evolved DNA aptamers from an oligonucleotide library binding to purified recombinant capsids but not binding to the corresponding I126A mutant capsids. Aptamers bound to capsids were separated from unbound molecules by filtration. After 13 rounds of selections and amplifications 16 different aptamers were found among 73 clones. The four most frequent aptamers represented more than 50 % of the clones. The main aptamer AO-01 (13 clones, 18 %) showed the lowest dissociation constant (Kd) of 180 +/- 82 nM for capsid binding among the four molecules. Its Kd value for I126A capsids was 1306 +/- 503 nM. Cotransfection of Huh7 cells with AO-01 and an HBV genomic construct resulted in 47 % inhibition of virion production 3 days post transfection but showed no inhibition by cotransfection of an aptamer with random sequence. The half-life of AO-01 in cells was 2 hours which might explain the incomplete inhibition. The results support the importance of the MBD for nucleocapsid envelopment. Inhibiting the MD-MBD interaction by a low molecular weight substance might represent a new approach for an antiviral therapy. IMPORTANCE: Approximately 240 million people are persistently infected with HBV. To date, antiviral therapies depend on a single target, the viral reverse transcriptase. Future additional targets could be viral protein-protein interactions. We selected a 55 base long single stranded DNA molecule (aptamer) which binds with relatively high affinity to a region on the HBV capsid interacting with viral envelope proteins during budding. This aptamer inhibits virion formation in cell culture. The result substantiates the current model for HBV morphogenesis and shows that the capsid envelope interaction is a potential antiviral target.