A novel atmospheric pressure matrix-assisted laser desorption ionization mass spectrometry (AP-MALDI-MS) method was established for the facile detection of pesticides in ambient environment. Four kinds of multi-walled carbon nanotubes (MWCNTs)-based matrix were synthesized and utilized to enhance the ionization efficiency of pesticides. Organophosphorus, anilinopyrimidine, carbamate, triazine, triazole and benzimidazole pesticides were directly desorbed and ionized from MWCNTs-based matrix in ambient environment, showing clear background and good sensitivity. In a comparison, Fe 3 O 4 -doped MWCNTs improved the intensity of pesticide ions more than the other three matrices. Moreover, MWCNTs-based matrix exhibited better performance than organic matrix. Quantitative analysis of pesticides using AP-MALDI-MS was validated to be adequate linearity, repeatability and sensitivity. Overall, AP-MALDI-MS combined with MWCNTs-based matrix enables the directly qualitative and quantitative analysis of pesticides in ambient environment. In this work, a novel atmospheric pressure matrix-assisted laser desorption ionization mass spectrometry (AP-MALDI-MS) method for the facile detection of pesticides was established.