Abstract: A dual-vector system for expressing an OTOF protein includes two nucleotide sequences, where a first nucleotide sequence includes two ITR sequences and a gene expression cassette inserted between the ITR sequences; a second nucleotide sequence includes two ITR sequences and a gene expression cassette inserted between the ITR sequences; the gene expression cassette of the first nucleotide sequence includes a promoter, an N-terminal coding sequence of OTOF, an N-terminal coding sequence of intein, and a polyA; and the gene expression cassette of the second nucleotide sequence includes a promoter, a C-terminal coding sequence of Intein, a C-terminal coding sequence of OTOF, and a polyA. The present disclosure further provides an adeno-associated virus packaged by the vector. The vector and the virus can recover hearing of bilateral ears by a unilateral ear administration in the field of deafness gene therapy large gene dual-vector delivery.