Graphene layers are potential candidates in a large number of applications. However, little is known about their ecotoxicological risks largely as a result of a lack of quantification techniques in complex environmental matrices. In this study, graphene was synthesized by means of graphitization and exfoliation of sandwich-like FePO4/dodecylamine hybrid nanosheets, and (14)C was incorporated in the synthesis. (14)C-labeled graphene was spiked to artificial freshwater and the uptake and depuration of graphene by Daphnia magna were assessed. After exposure for 24 h to a 250 μg/L solution of graphene, the graphene concentration in the organism was nearly 1% of the organism dry mass. These organisms excreted the graphene to clean artificial freshwater and achieved roughly constant body burdens after 24 h depuration periods regardless of the initial graphene exposure concentration. Addition of algae and humic acid to water during the depuration period resulted in release of a significant fraction (>90%) of the accumulated graphene, but some still remained in the organism. Accumulated graphene in adult Daphnia was likely transferred to the neonates. The uptake and elimination results provided here support the environmental risk assessment of graphene and the graphene quantification method is a powerful tool for additional studies.