The invention discloses a preparation method and an establishing method for a standard sample of transgenic rice flour BT63 as well as application of the standard sample in detection of the transgenic rice flour BT63. The preparation method comprises the following steps: respectively grinding and sieving the transgenic rice flour BT63 as a raw material; adding water and stirring to obtain lyophilized powder; and carrying out split charging and storing the lyophilized powder at the temperature of 0-4DEG C. The standard sample of the transgenic rice flour BT63, which is obtained by the method, can be also applied to a Real-Time PCR (Polymerase Chain Reaction) detection kit of the transgenic rice flour BT63 and has important real significance for comprehensively and deeply searching to solve a preparation technology and a stable ensuring technology of the standard sample for detecting transgenic components, actively developing the research on the standard sample for detecting transgenic products and filling up the blank of the measurement field.