The diamond back moth, Plutella xylostella, causes severe damage at all crop stages, beside its rising resistance to all insecticides. The objective of this study was to look for a new control strategy such as application of insecticide-loaded carbon dot-embedded fluorescent mesoporous silica nanoparticles (FL-SiO2 NPs). Two different-sized methoxyfenozide-loaded nanoparticles (Me@FL-SiO2 NPs-70 nm, Me@FL-SiO2 NPs-150 nm) were prepared, with loading content 15% and 16%. Methoxyfenozide was released constantly from Me@FL-SiO2 NPs only at specific optimum pH 7.5. The release of methoxyfenozide from Me@FL-SiO2 NPs was not observed other than this optimum pH, and therefore, we checked and controlled a single release condition to look out for the different particle sizes of insecticide-loaded NPs. This pH-responsive release pattern can find potential application in sustainable plant protection. Moreover, the lethal concentration of the LC50 value was 24 mg/L for methoxyfenozide (TC), 14 mg/L for Me@FL-SiO2 NPs-70 nm, and 15 mg/L for Me@FL-SiO2 NPs-150 nm after 72 h exposure, respectively. After calculating the LC50, the results predicted that Me@FL-SiO2 NPs-70 nm and Me@FL-SiO2 NPs-150 nm exhibited better insecticidal activity against P. xylostella than methoxyfenozide under the same concentrations of active ingredient applied. Moreover, the activities of detoxification enzymes of P. xylostella were suppressed by treatment with insecticide-loaded NPs, which showed that NPs could also be involved in reduction of enzymes. Furthermore, the entering of FL-SiO2 NPs into the midgut of P. xylostella was confirmed by confocal laser scanning microscope (CLSM). For comparison, P. xylostella under treatment with water as control was also observed under CLSM. The control exhibited no fluorescent signal, while the larvae treated with FL-SiO2 NPs showed strong fluorescence under a laser excitation wavelength of 448 nm. The reduced enzyme activities as well as higher cuticular penetration in insects indicate that the nano-based delivery system of insecticide could be potentially applied in insecticide resistance management.