The invention discloses double-copy human p53 gene recombinant adenovirus and a preparation method thereof. A commercialized 5-type recombinant replication-deficient adenovirus construction system (AdEasy) is inserted into a double-copy human p53 tumor inhibition gene eukaryotic expression box as shown in SEQ ID NO.1 to construct a p53 tumor inhibition gene recombinant adenovirus expression carrier system, and recombinant replication-deficient adenovirus granules for expressing double-copy human p53 tumor inhibition gene are further obtained. Experiment shows that after being injected by tumor cells, the double-copy human p53 gene recombinant adenovirus can efficiently express p53 tumor inhibition genes carried by the virus. As the double-copy human p53 tumor inhibition gene eukaryotic expression box is integrated, the p53 tumor inhibition gene expression amount can be greatly increased, meanwhile the virus amount can be reduced, and a relatively good gene treatment effect can be achieved. The double-copy human p53 gene recombinant adenovirus is good in specificity and wide in spectrum, directly aims at gene mutation of tumor cells, and can be applied to malignant tumor of various tissue types at the early stage, the middle stage and the late stage.