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Cerenkov luminescence imaging (CLI) has been widely investigated for biological imaging. However, the luminescence generated from Cerenkov effect is relatively weak and has poor penetration ability in biological tissues. These limitations consequently hindered the clinical translation of CLI. In this study, we proposed an in vitro experimental study for the demonstration of quantum dots (QDs) configurations affected by the improvement of the signal intensity of CLI. Results revealed that the optimal concentrations were 0.1 mg/mL and 0.25 mg/mL for the studied CdSe/ZnS QDs with fluorescence emission peaks of 580 nm and 660 nm, respectively. The detected optical signal intensity with long-wavelength emission QDs were stronger than those with short-wavelength emission QDs. This study illustrates an experiment to study the effects ofconcentrations and fluorescence emission peaks of QDs on an enhanced optical signal for the exteal detection of CLI.