The invention discloses a method for preparing a luminous bacteria freeze-dried preparation. The method specifically comprises the following steps: a, providing a liquid mixture containing luminous bacteria and a protective agent; b, cooling the mixture to a first temperature interval T1, and maintaining the first temperature interval T1 for a period of time P1; c, heating the cooled mixture from the first temperature interval T1 to a second temperature interval T2, and maintaining the second temperature interval for a period of time P2; d, heating the heated mixture from the second temperature interval to a third temperature interval T3, and maintaining the temperature for a period of time P3; and e, drying the dormancy mixture, thereby forming the luminous bacteria freeze-dried preparation. The luminous bacteria freeze-dried preparation prepared by the method disclosed by the invention is high in stability, and the effective time of the activity after recovery is long.