Abstract Purpose While immunotherapy has revolutionized the oncology field, variations in therapy responsiveness limit the broad applicability of these therapies. Diagnostic imaging of immune cell, and specifically CD8+ T cell, dynamics could allow early patient stratification and result in improved therapy efficacy and safety. In this study, we report the development of a nanobody-based immunotracer for non-invasive SPECT and PET imaging of human CD8+ T-cell dynamics. Methods Nanobodies targeting human CD8β were generated via llama immunizations and subsequent biopanning. The lead anti-human CD8β nanobody was characterized in vitro on binding, specificity, stability and toxicity. The lead nanobody was labelled with 99mTc and 68Ga for non-invasive imaging of human T-cell lymphomas and CD8+ T cells in human CD8 transgenic mice and non-human primates via SPECT or PET/CT. Repeated imaging of CD8+ T cells in MC38 tumor-bearing mice was performed to visualize CD8+ T-cell dynamics. Results The nanobody-based immunotracer showed high affinity and specific binding to human CD8 without unwanted immune activation. CD8+ T cells were non-invasively visualized via SPECT and PET imaging in naïve and tumor-bearing mice and in naïve non-human primates with high sensitivity. The nanobody-based immunotracer showed enhanced specificity for CD8+ T cells and/or faster in vivo pharmacokinetics compared to previous human CD8-targeting immunotracers, allowing us to follow human CD8+ T-cell dynamics already at early timepoints. Conclusion Overall, this study describes the development of a more specific human CD8+ T-cell-targeting immunotracer, allowing follow up of immunotherapy responses via non-invasive imaging of human CD8+ T-cell dynamics.