YSCH-01, a recombinant L-IFN oncolytic adenovirus based anticancer drug with dual anti-tumor effects, has displayed significant antitumor properties in cancer treatment. However, its specific role in osteosarcoma and the underlying mechanisms remain under studied. Therefore, we analyzed clinical samples and performed experiments using osteosarcoma cell lines in both in vitro and in vivo settings. At the RNA and protein levels, the expression of L-IFN gene carried by YSCH-01 showed the potential antitumor properties. RNA-seq analysis of tumor samples treated with YSCH-01 provided insights into the altered gene expression profiles and signaling pathways, shedding light on the molecular basis of its antitumor effects. Those findings demonstrated that YSCH-01 displayed dose-dependent infectivity with different levels of efficacy and a significant inhibition of osteosarcoma cell proliferation. In both cell line and patient derived xenograft models, YSCH-01 exhibited potent therapeutic effects with the activated type I interferon signaling pathway in tumor tissues, particularly in terms of its distant effects. Furthermore, this study found out the key signal pathways through which YSCH-01 exerts its antitumor effects in osteosarcoma, which pinpointed potential therapeutic targets by identifying the type I interferon signaling pathway as the candidate molecular markers and their correlation with patient outcomes, positioning YSCH-01 as a promising candidate for osteosarcoma treatment.