Gram-negative binding proteins (GNBPs) are important in the innate immune system of insects in recognition of fungi pathogen, such as Beauveria bassiana. However, this information in aphids is not clear, which might be exploited to develop a novel aphid control strategy based on integrating RNAi and B. bassiana. Here, we firstly identified two GNBPs, ApGNBP1 and ApGNBP2, using the model aphid Acyrthosiphon pisum, and observed that two ApGNBPs were highly expressed in hemolymph and fat body as well as upon the injection of β-1,3-glucan and heat-killed B. bassiana. Intriguingly, RNAi-based silencing of ApGNBP1 but not ApGNBP2 decreased the activity of immune-related phenoloxidase. This led to the increased virulence of B. bassiana in A. pisum upon silencing of ApGNBP1, and the synergetic effects were also observed in other two aphids: Myzus persicae and Aphis citricidus. Importantly, no negative effects were detected in aphid predator Propylaea japonica under the co-application of the RNAi in targeting ApGNBP1 and B. bassiana. Taking together, we conclude that GNBP1 could be an optimal target in aphid control by combining RNAi, entomopathogenic fungi and ladybeetle predator.