Rabies is a zoonotic infectious disease caused by rabies virus (RABV), and its mortality rate is as high as 100%. Globally, an average of 60,000 people die from rabies each year. The most effective method to prevent and limit rabies is vaccination, but it is currently expensive and inefficient, consisting of a 3-dose series of injections and requiring to be immunized annually. Therefore, it is urgent to develop a single dose of long-acting rabies vaccine. In this study, recombinant rabies virus (rRABV) overexpressing interleukin-33 (IL-33) was constructed and designated as rLBNSE-IL33, and its effect was evaluated in a mouse model. The results showed that rLBNSE-IL33 could enhance the quick production of RABV-induced immune antibodies as early as three days post immunization (dpi) through the activation of dendritic cells (DCs), a component of the innate immune system. Furthermore, rLBNSE-IL33 induced high-level virus-neutralizing antibodies (VNA) production that persisted for 8 weeks by regulating the T cell-dependent germinal center (GC) reaction, thus resulting in better protection against rabies. Our data suggest the IL-33 is a novel adjuvant that could be used to enhance innate and humoral immune responses by activating the DC-GC reaction, and thus, rLBNSE-IL33 could be developed as a safe and effective vaccine for animals.