Background <p>Inducible T-cell co-stimulator receptor (ICOS) is a conserved biomarker mainly expressed on activated T cells. It had been proved that PET imaging targeting ICOS was a promising strategy for assessment of immune responses in cancer immunotherapy. For further translating this strategy into clinic, in the present study, we had developed the first-in-class ICOS targeting nanobody ICOS-53 via alpaca immunization and yeast display screening.</p> Results <p>The binding affinity of ICOS-53 to recombinant ICOS protein was validated by surface plasmon resonance, and the KD value was 85.3 pM. ICOS-53 was then conjugated to NOTA and radiolabeled with [⁶⁸Ga]GaCl₃. In PET imaging study, higher accumulation of [⁶⁸Ga]Ga-NOTA-ICOS-53 could be observed in CHO-ICOS (CHO transfected by human ICOS) tumors, compared to CHO (Chinese Hamster Ovary cells) cohort from all-time points examined. Statistically significant correlation between PET imaging quantitative results and biodistribution could be observed (<i>R</i> = 0.548, <i>P</i> = 0.00117). Immunofluorescence staining also confirmed the high ICOS expression in CHO-ICOS tumors.</p> Conclusion <p>Our data demonstrated that we had developed a novel nanobody based PET tracer targeting human ICOS, [⁶⁸Ga]Ga-NOTA-ICOS-53 PET imaging was a promising strategy for tracking ICOS+ cells both in vitro and in vivo.</p>