The invention discloses a haliotis discus hannai microsatellite molecular marker which can analyze the genetic diversity of haliotis discus hannai wild population and identify a genetic relationship between cultured stocks and a preparation method thereof. According to the steps of establishing a haliotis discus hannai microsatellite (AC)n library, screening microsatellite sequence positive clone, designing a microsatellite primer, performing polymerase chain reaction (PCR) amplification and screening, ten highly polymorphic microsatellite molecular markers of Hd1, Hd2, Hd3, Hd4, Hd5, Hd6, Hd7, Hd8, Hd9 and Hd10 are obtained; and the polymorphic detection proves that the microsatellite molecular marker has the characteristic of high repeatability, is an effective and reliable molecular marker, can establish a haliotis discus hannai microsatellite DNA marker technology system and is used for evaluating the genetic diversity of haliotis discus hannai, analyzing the genetic structure and identifying the genetic relationship between the cultured stocks.