Enterobactin (Ent)-mediated high-affinity iron acquisition is critical for Gram-negative bacteria to survive in the host. Given the bacteriostatic effect of lipocalin resulting from its potent Ent-binding ability, immune intervention directly targeting Ent is promising for iron-dependent pathogen control. Recently, an Ent conjugate vaccine was reported, but it still has several significant weaknesses. In this study, we sought to develop an innovative Ent conjugate vaccine that can induce a high level of antibodies directed against Ent and to provide solid evidence demonstrating siderophore-binding capacity of Ent-specific antibodies. Using a simple method, we successfully conjugated purified Ent to different carriers, including keyhole limpet hemocyanin (KLH), bovine serum albumin, and CmeC, a vaccine candidate for Campylobacter control. Subcutaneous immunization of rabbits with the KLH-Ent conjugate triggered a strong systemic IgG immune response with an up to 16,384-fold increase in IgG titer directed against whole conjugate and an up to 4,096-fold increase in the level of specific anti-Ent IgG. To evaluate the ability of Ent-specific IgG to bind to the Ent derivatives present in vivo, various Ent derivatives were chemically synthesized and a unique enzyme-linked immunosorbent assay method was developed. The Ent-specific IgG also displayed exceptional reactivity to ferric Ent, a linear trimer of Ent, and different salmochelins. Growth assays further demonstrated that the Ent-specific antibodies significantly inhibited Ent-dependent growth of Campylobacter spp. and Escherichia coli Collectively, this study reports an efficient method to prepare a new type of Ent conjugate vaccines for inducing a high level of Ent-specific antibodies, which can bind to various Ent derivatives and display lipocalin-like bacteriostatic features.IMPORTANCE Ent-mediated high-affinity iron acquisition is a universal and critical contributor for Gram-negative pathogens to survive and infect hosts. Published information has supported an innovative immune intervention strategy that directly targets Ent to starve pathogens by limiting the availability of iron to be utilized. Compared to a recently published Ent conjugate, there are three advantages of the vaccine described in this study: ease of preparation, induction of high titer of anti-Ent IgG, and the ability of Ent-specific antibodies to bind various Ent derivatives, including the salmochelins that help enteric pathogens evade sequestration of siderophores by host lipocalins. In addition, the Ent-specific antibodies were demonstrated to function similarly to lipocalin to interfere with the Ent-dependent growth of Campylobacter and E. coli under iron-restricted conditions. This study has significant potential for broader applications to prevent and control various Gram-negative infections in humans and animals.