Abstract: Small molecular compounds can be analyzed by atmospheric pressure matrix-assisted laser desorption ionization mass spectrometry (AP-MALDI MS) using multi-walled carbon nanotubes (MWCNTs) as matrix. Three kinds of functionalized MWCNTs including oxidized MWCNTs, Fe₃O₄-doped MWCNTs and β-cyclodextrin-coated MWCNTs were synthesized, which together with intrinsic MWCNTs, were employed as matrixes to facilitate the ambient ionization of small molecular compounds without matrix interference. Six amino acids and three alkaloids of proline, DL-methionine, isoleucine, histidine, glutamic acid, aspartic acid, berberine hydrochloride, harmane and norharmane were studied to evaluate the difference on the ionization between MWCNTs-based matrix and traditional organic matrix, as well as the effects of experimental conditions on the MS peak noise and intensity. As a result, all the MWCNTs-based matrixes exhibited no matrix interference in the region of low mass-to-charge ratio, and could effectively promote the ionization of small molecular compounds. All the detected amino acids and alkaloids presented a soft ionization under the laser radiation using these matrixes, the fragmentation of the analytes were avoided, and the quasi-molecular ions were steadily obtained in their M+H⁺ or M+alkali⁺ formats. Four kinds of methods for sample preparation were optimized to enhance the ionization efficiency as well as the ion abundance. It was shown that the matrix-first method was best for the ionization of amino acids and alkaloids. The performances of the four MWCNTs-based matrixes on the assisted ionization were further evaluated. β-Cyclodextrin-coated MWCNTs was superior to the others in increasing the MS peak intensity and reducing the background noise. Using the functionalized MWNCTs as matrix, AP-MALDI MS showed good repeatability and sensitivity to determine the amino acids and alkaloids. The correlation coefficients R² of all the samples in five different concentrations were no less than 0.996 9 in the range of 10.0-300 mg/L. The relative standard deviations (RSDs) of in-spot repeatability were lower than 4.6%, while the spot-to-spot repeatability RSDs were lower than 6.7%. In a word, the AP-MALDI MS method combined with MWCNTs-based matrixes circumvents the matrix interference of conventional organic matrixes, and enables the directly facile analysis of small molecular compounds in ambient environment with reduced analysis time and simplified sample preparation. This work may provide a new way to rapid qualitative and quantitative analysis of small molecular compounds.