Gas-phase N-doped graphene (gNG) was synthesized by a modified thermal annealing method using gaseous melamine as nitrogen source and then for the first time applied as a matrix in negative ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for small molecule analysis. Unlike the complicated adducts produced in positive ion mode, MS spectra obtained on gNG matrix in negative ion mode was only featured by deprotonated molecule ion peaks without matrix interference. By the gNG assisted desorption/ionization (D/I) process, some applications were carried out on a wide range of low-molecular weight (MW) analytes including amino acids, fatty acids, peptides, anabolic androgenic steroids as well as anticancer drugs, with an extraordinary laser desorption/ionization (LDI) efficiency over traditional α-cyano-4-hydroxycinnamic acid (CHCA) and other carbon-based materials in the negative ion detection mode. By comparison of a series of graphene-based matrixes, two main factors of matrix gNG were unveiled to play a decisive role in assisting negative ion D/I process: a well-ordered π-conjugated system for laser absorption and energy transfer; pyridinic-doped nitrogen species functioning as deprotonation sites for proton capture on negative ionization. The good salt tolerance and high sensitivity allowed further therapeutic monitoring of anticancer drug nilotinib in the spiked human serum, a real case of biology. Signal response was definitely obtained between 1 mM and 1 μM, meeting the demand of assessing drug level in the patient serum. This work creates a new application branch for nitrogen-doped graphene and provides an alternative solution for small molecule analysis.