The invention discloses a construction method of a tumor radiotherapy virulent gene mutation library. The method is characterized in that totally 61 inheritable variation points on 42 human tumor radiotherapy virulent genes are covered. The construction method has the advantages that single management is performed on various target sequences, the construction of the library is rapidly accomplished, the construction process of the whole library only requires 2 to 3 hours, the manual time only requires 45 minutes, the present problem that multiple genes and target points are required to be detected when the condition that whether quite big toxic and side effects can be generated or not is predicated before a patient receives clinic tumor radiotherapy is effectively solved, and the cost is low.