The invention provides a construction method of targeting wnt signal pathway oncolytic adenovirus. The method comprises the following steps: 1, inserting a promoter into pXC2-delta24, connecting the promoter inserted pXC2-delta24 to a plasmid, and carrying out enzyme digestion; 2, connecting TSLC1 gene with the plasmid, mixing the obtained connection product with DH5alpha competent cells, and carrying out enzyme digestion; 3, connecting a final product obtained in step 1 with a final product obtained in step 2, transforming the obtained connected product into complete sequence adenovirus skeleton-containing plasmid Escherichia coli, and carrying out recombination to generate an adenovirus skeleton DNA plasmid missing the 24 bases of an E1A-CR2 region and substituting an E1A endogenous promoter in an the E1A region by the promoter; linearly transforming the adenovirus skeleton DNA plasmid by using pacI enzyme digestion, transferring the transformed adenovirus skeleton DNA plasmid into 293A cells, and carrying out virus packaging and pathological change to obtain the oncolytic adenovirus Ad.wnt-E1A(delta24)-TSLC1. The oncolytic adenovirus obtained by adopting the construction method has an obvious killing effect on colorectal carcinoma cells and liver cancer cells.