By using I-V, EL-V, displacement current measurement (DCM) and time-resolved electric-field-induced optical second-harmonic generation (TR-EFISHG) measurement, we studied the influence of interface pentacene layer inserted between ITO and a-NPD layers in ITO/α-NPD/Alq3/Al OLEDs. All experiments were carried out for the OLEDs with and without a pentacene interface layer. The I-V and EL-V measurements showed the decrease of operating voltage of EL, the DCM showed the lowering of inception voltage of carrier injection by inserting a pentacene interface layer. The TR-EFISHG measurement showed the faster accumulation of holes at the interface between the a-NPD and Alq3 layers, which resulted in the relaxation of electric field of a-NPD layer accomplished by the increase of the conductivity and the increase of the electric field in the Alq3 layer. We conclude that TR-EFISHG measurement is helpful for understanding I-V and EL-V characteristics, and can be combined with other methods to give significant information which are impacted by the interface layer.