A binary system composed of carbon dots (CDs) and N-doped CDs (N-CDs) embedded in an organic matrix was used for the analysis of cholesterol by MALDI (matrix-assisted laser desorption and ionization time-of-flight) mass spectrometry, as a model for detection of small, biologically relevant molecules. The results showed that both CDs are sensitive to the cholesterol and can be used either alone or in a binary system with 2,5-dihydroxybenzoic acid (DHB) to enhance the detection process. It was found that both COOH and NH2 groups on CDs surface contributed to the enhancement in the cholesterol detection by MALDI mass spectrometry in the presence of inorganic cations. Nevertheless, in the presence of NaCl, N-CDs led to a better reproducibility of results. It was due to the coexistence of positive and negative charge on N-CDs surface that led to a homogeneous analyte/substrate distribution, which is an important detection parameter. The enhancing effect of carbon dots was linked to a negative Gibbs energy of the complex formation between CDs, Na+, cholesterol and DHB, and it was supported by theoretical calculations. Moreover, upon the addition of CDs/N-CDs, such features as a low ionization potential, vertical excitation, dipole moment and oscillator strength positively affected the cholesterol detection by MALDI in the presence of Na+. (C) 2021 Elsevier Inc. All rights reserved.