Tetraspanin proteins are closely associated with high-curvature membrane structures and play key roles in organizing membrane domains and regulating membrane signaling in immune cells. However, their specific roles in regulating T cell membrane signaling, particularly within the microvilli often characteristic of these cells, remain poorly understood. Here, we used Jurkat T cells as a model system and investigated CD81 as a member of the tetraspanin family. Using total internal reflection fluorescence (TIRF) microscopy and structured illumination microscopy (SIM), we identified an enrichment of the tetraspanin CD81 microdomains along the actin-rich membrane microvilli. At the distal end of the microvilli, SIM images revealed the spatial colocalization of CD81 with T cell receptors (TCR) and CD63, implying a potential role for CD81 in regulating TCR signaling in conjunction with CD63. Spatial analysis of CD81 and CD63 microdomains from the dual-color SIM data revealed their preference for associating with each other. Cluster analysis of direct stochastic optical reconstruction microscopy (dSTORM) data revealed that in vitro T cell activation results in reduced domain sizes and increased domain separation of CD81. These findings provide visual evidence of the spatial organization and rearrangement of CD81 on the T cell microvilli, highlighting its potential role in signal regulation on specialized membrane protrusions.