Abstract Background Tertiary lymphoid structures (TLS) are ectopic lymphoid formations within or around tumours, emerging as important predictors of prognosis and response to immune checkpoint inhibitors (ICI) in various cancers. However, in Merkel cell carcinoma (MCC), the correlation between TLS and ICI response has not yet been evaluated. Furthermore, the criteria for assessing TLS maturity vary by report, and there is no unified consensus. Objective In this study, we investigated the relationship between TLS and ICI responsiveness in MCC using spatial proteomics at the single-cell level. By comparing the spatial characteristics of TLS in ICI responders and non-responders, we also aimed to identify features related to functionality of TLS, distinct from the previously reported maturity of TLS. Methods We analysed 24 TLSs from 13 MCC cases treated with the anti-PD-L1 antibody, avelumab, employing the PhenoCycler platform with 16 oligonucleotide-labeled antibodies. Infiltrating immune cells and tumour cells were phenotyped based on machine learning, and spatial single-cell analysis was performed, including cellular neighbourhood analysis and cell-cell correlation analysis. Results Our findings revealed that the number of TLS positively correlated with survival and ICI response (p=0.0274). The number of TLS observed within the tumour was significantly higher in responders than in non-responders (p=0.0043). Cellular neighbourhood analysis and cell-cell correlation analysis demonstrated that in functional TLS in responder cases, T cells infiltrated densely within B cell clusters, extending close to follicular dendritic cells, and CD4-positive T cells adjacent to CD8-positive T cells express PD1. In contrast, in non-responder TLS, although B cell density is high, T cells did not infiltrate into B cell cluster areas but instead resided in the peripheral areas. Furthermore, it was found that CD8-positive T cells and CD4-positive T cells approaching B cells expressed PD1. Conclusions This study demonstrated a significant positive correlation between the number of TLS and ICI response in MCC. Additionally, our results suggest that the immune composition and spatial configuration within TLS may be important determinants of ICI efficacy.