A green and facile method was developed by synthesizing a cuprous oxide–carbon dots/Nafion (Cu₂O–CDs/NF) composite film for highly sensitive and reliable determination of dopamine (DA). The Cu₂O nanoparticles could improve the conductivity of the electrode, while the CDs with carboxyl groups and the NF with sulfo groups could attract cations via the ion-exchange model and exclude anions by the electrostatic action. The proposed biosensor exhibited a low detection limit of 1.1 nM with a wide linear range of 0.05–45.0 μM and acquired excellent sensitivity and selectivity for DA. Furthermore, the Cu₂O–CDs/NF/GCE also was applied to determine DA in human serum with satisfactory results and showed good activity for more than two months.