Biomass-derived carbon dots were synthesized by a hydrothermal method using the flavonoid luteolin, 3 0 ,4 0 ,5,7-tetrahydroxyflavone, as a carbon source. The reaction conditions were optimized with regard to reaction temperature, reaction time, and amount of luteolin. N-doped luteolin-based carbon dots (N-CDs) were obtained through the addition of ethylenediamine and used as MALDI-TOF matrix for the analysis of amino acids and other low molecularweight molecules. Compared with N-free CDs, the matrix application of N-CDs displayed less background interferences, high sensitivity, superior efficiency, and good repeatability. Pyridinic N atoms were identified as the major component of the nitrogen dopants in N-CDs. The lone electron pair of this N species, which is located in an sp 2 -orbital perpendicular to the aromatic system, and the π-conjugated structure contributed to an enhanced desorption/ ionization efficiency. Furthermore, the qualitative detection of amino acids in serum samples was accomplished by MALDI-TOF MS using the N-CD matrix. This new type of MALDI-TOF matrix demonstrated its potential in the analysis of small molecules present in chemical, environmental, or biological samples.