The photocurrent generated from chloroplasts of spinach is investigated using a carbon paste electrode. Chloroplasts extracted from spinach are shown to interact with a carbon paste electrode through five types of redox mediators. Among these mediators, 2,5-dichloro-1,4-benzoquinone (2,5-DCBQ) is found to be the best electron-transfer mediator, generating the highest photocurrent of approximately 310 nA cm−2 per μg chlorophyll. In addition, the photocurrent density is investigated as a function of the photon flux density, concentration of 2,5-DCBQ, and the amount of chloroplasts deposited onto the surface of the electrode. Photoreduction of 2,5-DCBQ exhibited Michaelis–Menten-like kinetics, and the reduction rates were dependent on the amount of 2,5-DCBQ and the light intensity. Even 10 days after the chloroplasts were extracted, the electrodes with the extracted spinach chloroplasts exhibited ∼90% of the photoinduced current that they exhibited immediately after the chloroplast extraction. The source of photocurrent was confirmed in experiments using 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of electron transfer from primary quinone electron acceptor QA to secondary quinone electron acceptor QB in photosystem II, and KCN, an inhibitor of electron transfer from cytochrome b6f complex to photosystem I.