The invention relates to a gene chip for non-invasive prenatal diagnosis of high-risk hereditary hearing loss and a preparation method in the technical field of gene diagnosis. The gene chip comprises a substrate and probes fixed on the substrate, wherein the probes are nucleotide sequences shown as SEQ ID NO.1-24. Usage of the gene chip includes the steps of sample preparation, multiplex PCR (polymerase chain reaction) amplification, ligase detection reaction, chip hybridization, chip scanning and result obtaining. Beside, the invention further relates to a reagent kit comprising the gene chip. The gene chip for diagnosis is simple in operation steps, only requires the multiplex PCR reaction, the ligase detection reaction and hybrid scanning once, is high in detection specificity and good in stability so as to be capable of correctly distinguishing homozygotes from heterozygotes of each locus and high in repeatability of repeated experiments, is short in detection time and low in cost as the substrate can be universally used for other chips by means of universal chip technology.