Objective Recombinant adeno-associated virus expressing circKat6b（rAVV-circKat6b）was obtained and its infection effect and expression level of circKat6b in the hippocampus of mice were examined. Methods The mmu-circKat6b gene fragment was inserted into AAV expression plasmid to construct a recombinant AAV plasmid. The recombinant AAV plasmid，packaging plasmid and helper plasmid were co-transfected into 293T cells by LipofiterTM for rAAV-circKat6b packaging. The rAAV-circKat6b was collected from transfected 293T cells and purified by column，and the titer of the rAAV-circKat6b was tested by qRT-PCR. Finally， rAAV-circKat6b was injected into the hippocampus of mice by stereotactic apparatus. One month later，the infection effect of rAAVcircKat6b was observed by immunofluorescence microscope，and the expression level of rAAV-circKat6b was detected by qRT-PCR. Results The successfully packaged rAAV-circKat6b was purified to obtain high titer rAAV-circKat6b，and the virus titer of rAAVcircKat6b was detected by qRT-PCR and reached 1.2×1012 vg/mL. Strong green fluorescence and high level of circKat6b were observed in the infected hippocampus after one month of infection. Conclusions The successfully constructed rAAV-circKat6b could highly increase the expression level of circKat6b in the hippocampus of mice，which provided an effective tool for further studying of the biological functions of circKat6b.