Ambipolar transport is crucial for constructing high performance organic light-emitting transistors (OLETs), but the ambipolar feature is usually not exhibited due to ineffective electron injection especially in symmetric device geometry. Herein, we show that electron injection could be greatly enhanced through the judicious design of an organic interface layer of 3,7-di(2-naphthyl)dibenzothiophene S,S-dioxide (DNaDBSO) which shows an interfacial dipole effect upon contact with a metal electrode, especially an Au electrode. When incorporating a DNaDBSO film beneath Au electrodes, the electron injection and mobility were significantly enhanced in 2,6-diphenylanthracene-based OLETs, and thus ambipolar transport (μmaxh: 2.17 cm2 V-1 s-1, μmaxe: 0.053 cm2 V-1 s-1) was effortlessly obtained. Furthermore, the shift of the electroluminescent region was obviously observed upon modulation of gate voltage, which demonstrates efficient electron injection and intrinsic ambipolar transporting properties in devices. This study provides a new avenue for regulating the interface in electroluminescent devices towards high performance simple-structured OLETs in applications.