Abstract: Ultra-high performance concrete (UHPC) has excellent mechanical and impermeability properties, and has a broad application prospect in the concrete structure reinforcement project. How to improve the bonding performance between UHPC and existing substrate has become an important topic of general concern in the field of civil engineering. In this paper, experimental studies were carried out using specimens prepared under standard curing conditions, and the effects of the water-binder ratio (w/b) of UHPC and the initial water saturation of the concrete substrate on the interfacial shear strength between them were first investigated. In addition, the evolution law of interfacial shear strength with age between the selected UHPC with a specific mix proportion and concrete with different saturation was investigated. Pre-wetted lightweight aggregate was incorporated into UHPC to achieve internal curing, and the evolution law of the interfacial shear strength between lightweight aggregate UHPC and concrete substrate after curing with different ages was investigated by taking into account the effects of the replacement rate of lightweight aggregate in UHPC, and the water saturation of concrete substrate. The microstructure of the interface between UHPC and concrete was observed using scanning electron microscopy. The results show that (1) for UHPC without lightweight aggregates, the interfacial shear strength between it and water-saturated substrate is the highest while the interfacial shear strength between it and dry substrate is the lowest irrespective of the change of the w/b of UHPC. The interfacial shear strength between UHPC and dry substrate decreases with the increase of w/b, and that between UHPC and pre-wetted substrate increases first and then decreases with the increase of the w/b. (2) The interfacial shear strengths between UHPC with a w/b of 0.154 and different saturated substrate all increase significantly at 28 d compared to that at 7 d. At 90 d, only the interfacial shear strengths between it and 50% saturated substrate further increase significantly. (3) A low lightweight aggregate replacement rate can increase the interfacial shear strength between UHPC and dry substrate at 7 d, while a high lightweight aggregate replacement rate can significantly increase the interfacial shear strength between UHPC and dry substrate at 28 d and 90 d. The interfacial shear strength between lightweight aggregate UHPC and 50% saturated substrate is significantly higher than that between lightweight aggregate UHPC and dry substrate at 28 d and 90 d. At 7 d, a certain interfacial shear strength can be formed between UHPC with different lightweight replacement rates and water-saturated substrate, and the strength further increases at both 28 d and 90 d.