Provided are an Ebola virus envelope glycoprotein (that is GP protein) codon optimized nucleotide sequence, a human replication deficient recombinant adenovirus capable of expressing the nucleotide sequence, and applications in preparing a vaccine for preventing Ebola virus diseases. The nucleotide sequence takes a replication deficient 5 type adenovirus that is lack of E1 and E3 in a combined mode as a vector. HEK293 cells that integrate adenovirus E1 genes serve as a packaging cell line, and carried protective antigenic genes are codon optimized Zaire type Ebola virus Makona strain envelope glycoprotein genes. After the envelope glycoprotein genes are optimized by codon, the expression level in transfection cells is obviously improved.