By using electric-field-induced second-harmonic generation (EFISHG) measurement, we analyzed photovoltaic effect of two-layer solar cells (indium zinc oxide/pentacene/C60/Al). Results evidently showed that negative and excessive charges Qs accumulated at the two-layer interface under illumination, e.g., Qs =-1.7×10-9 C/cm2 at 0.05 mW/cm2 and –3.6×10-9 C/cm2 at 0.5 mW/cm2, while a short-circuit current flowed. The open-circuit voltage changed in accordance with accumulation charge Qs, and finally saturated. Modeling that accumulated negative charge is a source of space charge field and directly effects on the electrostatic energy stored in OSCs, dependence of the open voltage on the accumulated charge Qs was explained.