Amaranthus palmeri is one of the most troublesome invasive agricultural weeds worldwide, exhibiting super invasiveness and high resistance to conventional management strategies. Artificial microRNA-mediated silencing technology, coupled with a nanoparticle-mediated delivery system, represents an attractive approach for fertility control in A. palmeri. In this study, we first characterised the biological function of ApMIR319 via ectopic overexpression in Arabidopsis, identifying it as a crucial candidate molecular target for fertility regulation in A. palmeri. Subsequently, we prepared layered double hydroxide (LDH) nanosheets using the co-precipitation-hydrothermal method. Employing the LDH nanosheets as nanocarriers, we implemented nearly complete encapsulation of the ApmiR319 mimic at a mass ratio of 1:100. The LDH-ApmiR319 mimic complex exhibited stable loading capacity in neutral and alkaline solutions. Furthermore, the LDH-ApmiR319 mimic complex demonstrated robust adhesion to leaf surfaces and enhanced resistance to enzymatic degradation. Spraying treatments with the LDH-ApmiR319 mimic complex significantly elevated ApmiR319 expression levels in male florets, while concurrently down-regulating its target genes (ApTCP4, ApTCP10 and ApMYB33), thereby inhibiting pollen development in A. palmeri. In conclusion, this study successfully established an LDH nanosheet-mediated delivery system of ApmiR319 mimic for male fertility control in A. palmeri. It represents a novel strategy and direction for achieving sustainable management of this weed.