The invention provides a preparation method of tortoise-shell glue. The preparation method comprises the following steps: (1) thoroughly cleaning a tortoise-shell and crushing the tortoise-shell for later use; (2) putting the crushed tortoise-shell in a container to be dry-steamed; (3) adding water into the dry-steamed container in the step (2) to be cooked, drained and cleaned; (4) adding water into the steamed tortoise-shell and pressurizing the tortoise-shell to be fully cooked to prepare a mixed sample of a tortoise-shell body solution; (5) filtering and separating the mixed sample to obtain a glue solution; and (6) concentrating the glue solution to obtain a tortoise-shell glue product. In a preparation process, the content of volatile basic nitrogen is effectively controlled, so that the tortoise-shell glue is high in safety, effectively treats various diseases and is relatively wide in application prospect.