The rise of programmed death-1 (PD-1)/PD-L1 immune checkpoint inhibitor therapy has been one of the most promising developments in melanoma research. However, not all the melanoma patients respond to such immune checkpoint blockade. There is a great need of biomarkers for appropriate melanoma patient selection and therapeutic efficacy monitoring. The objective of this study is to develop a novel radiolabeled anti-PD-L1 antibody fragment, as an imaging biomarker, for evaluating the PD-L1 levels in melanoma. The Df-conjugated F(ab’) fragment of the anti-mouse PD-L1 antibody was successfully synthesized and radiolabeled with Zr. Both Df-F(ab’) and Zr-Df-F(ab’) maintained the nano-molar murine PD-L1 targeting specificity and affinity. Zr-Df-F(ab’) showed less uptake in normal liver tissue in mice compared with its full antibody counterpart Zr-Df-anti-PD-L1. Positron emission tomography (PET)/computed tomography images clearly showed that Zr-Df-F(ab’) possessed superior pharmacokinetics and imaging contrast over the radiolabeled full antibody, with much earlier and higher tumor uptake (5.5 times more at 2 h post injection) and much lower liver background (51% reduction at 2 h post injection). The specific and high murine PD-L1-targeting uptake at tumor foci coupled with fast clearance of Zr-Df-F(ab’) highlighted its potential for PET imaging of murine PD-L1 levels and future development of radiolabeled anti-human PD-L1 fragment for potential application in melanoma patients.