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Human adenoviruses (HAdVs) are major respiratory pathogens. Specifically, human adenovirus type 4 (HAdV4) and human adenovirus type 7 (HAdV7) are known for causing fever and pneumonia, with documented cases of fatalities among the population. In recent years, HAdV4/HAdV7 has been implicated in causing substantial outbreaks, leading to increased morbidity in multiple countries. Most HAdV4 and HAdV7 infections have been reported in North America, Asia, Europe, Africa, South America, Oceania, and the Middle East. Most fatalities occurred in North America (the United States) and Asia (China and Singapore). Engineered recombinant adenoviruses have played a crucial role as vaccine vectors. In this study, we constructed a recombinant adenovirus, Ad4ITRmut-Ad7E3, and evaluated it in vitro and in vivo. We observed that the replication rate of Ad4ITRmut-Ad7E3 was lower than that of the RI-67 strain, indicating that the mutation of inverted terminal repeats (ITRs) weakened the replication ability of HAdV4. Immunization of BALB/c mice with the bivalent Ad4ITRmut-Ad7E3 vaccine strain, administered by intraperitoneal injection and oral gavage, resulted in the elicitation of neutralizing antibodies targeting HAdV4 and HAdV7. This finding not only provides a novel method and technique for the efficient construction of a polyvalent recombinant adenovirus vaccine candidate against HAdV4 and HAdV7 but also against other prevalent adenovirus serotypes such as HAdV3, HAdV11, HAdV14, and HAdV55, from various regions.