Background: Tumor infiltrating lymphocytes (TILs), the main component in the tumor microenvironment, play a critical role in the antitumor immune response. Few studies have developed a prognostic model based on TILs in osteosarcoma. Methods: ScRNA-seq data was obtained from our previous research and bulk RNA transcriptome data was from TARGET database. WGCNA was used to obtain the immune-related gene modules. Subsequently, we applied LASSO regression analysis and SVM algorithm to construct a prognostic model based on TILs marker genes. What's more, the prognostic model was verified by external datasets and experiment in vitro. Results: Eleven cell clusters and 2044 TILs marker genes were identified. WGCNA results showed that 545 TILs marker genes were the most strongly related with immune. Subsequently, a risk model including 5 genes was developed. We found that the survival rate was higher in the low-risk group and the risk model could be used as an independent prognostic factor. Meanwhile, high-risk patients had a lower abundance of immune cell infiltration and many immune checkpoint genes were highly expressed in the low-risk group. The prognostic model was also demonstrated to be a good predictive capacity in external datasets. The result of RT-qPCR indicated that these 5 genes have differential expression which accorded with the predicting outcomes. Conclusions: This study developed a new molecular signature based on TILs marker genes, which is very effective in predicting OS prognosis and immunotherapy response. Keywords: immunology; osteosarcoma; risk score; single-cell RNA sequencing; tumor infiltrating lymphocytes.