Lung squamous cell carcinoma (LSCC) is refractory to various therapies for non-small cell cancer; therefore, new therapeutic approaches are required to improve the prognosis of LSCC. Although immunotherapies targeting B7 family molecules were explored as treatments for several cancer types, the expression and significance of B7-H3 in the tumor microenvironment (TME) and its relationship with other immune checkpoint molecules have not yet been investigated in detail. We used high-throughput quantitative multiplex immunohistochemistry to examine B7-H3 expression in the TME. We investigated the relationship between B7-H3 expression and prognosis as well as changes in the TME with B7-H3 expression using 110 surgically resected pathological specimens retrospectively. We examined the correlation between B7-H3 and programmed cell death-ligand 1 (PD-L1) expression in single cells. High B7-H3 expression in tumor cells was associated with a better prognosis and a significant increase in the number of CD163PD-L1 macrophages. Quantitative analysis revealed that there is a positive correlation between B7-H3 and PD-L1 expression in tumor and stromal cells, as well as in intratumoral tumor-infiltrating lymphocytes and tumor-associated macrophages in the same cells. CD68, CD163, and CK cells with PD-L1 phenotypes had higher B7-H3 expression compared to PD-L1 cells. Our findings demonstrate a correlation between B7-H3 and PD-L1 expression in the same cells, indicating that therapies targeting B7-H3 could provide additional efficacy in patients refractory to PD-L1-targeting therapies.