It is still a challenge to improve ionization efficiency of saccharides in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Herein, the highly curved onion-like carbon nanoparticles (OCS) were synthesized from the low-price candle raw via a facile strategy. The unique nanostructure of OCS showed large surface area with plentiful mesoporous architecture, highly curved sp2 carbon with regulating electronic effect, and good hydrophilicity, which could be beneficial to facilitate the desorption and ionization efficiency in MS process. The prepared OCS material as MALDI matrix exhibited the superior performance for the detection of xylose, glucose, maltose monohydrate, and raffinose pentahydrate in positive-ion mode with low background noise, enhanced ion intensities, uniform distribution, excellent reproducibility, good salt-tolerance, and high sensitivity compared to control candle soot (CS) and traditional α-cyano-4-hydroxycinnamic acid (CHCA) matrices. This highly effective LDI of OCS matrix was attributed to its enhancing local electric field effect, strong UV absorption ability, and high photo-thermal conversion performance. Furthermore, the OCS-assisted LDI MS approach was employed to quantitatively detect glucose in rat serum. This LDI MS platform may have valuable for the analysis of metabolites in clinical research.