Graphene oxide (GO), as a promising synergistic agent of pesticides against pests, exhibits broad application prospects in the agricultural field. However, the synergistic mode of GO on acaricides remains unclear. Herein, an acaricide delivery nanosystem using GO nanosheets as nanocarriers, for the first time, was constructed and characterized to deliver four global top-selling acaricides (avermectin, bifenazate, etoxazole and spirodiclofen) to control Tetranychus cinnabarinus (a crop-threatening spider mite pest). The results showed that GO adsorbed on and impaired the cuticle of mites by binding to a cuticle protein (CPR) and inhibiting the expression of the CPR gene, thereby increasing the cuticle permeability of mites, which significantly enhanced the efficiency of acaricides. Additionally, silencing the CPR gene by RNAi resulted in dehydration, disturbed the construction of the cuticle layer, and increased the cuticle permeability and sensitivity of mites to GO–acaricides, which was consistent with the synergistic phenotype of GO on acaricides, demonstrating that the molecular mechanism of synergistic effects of GO on acaricides against T. cinnabarinus was mediated by the low-expressed CPR gene. The use of GO–acaricides in agriculture will reduce the application of chemical acaricides and their adverse impacts on public health and the environment.