Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has been widely used for biomolecular analysis. However, with conventional MALDI, it is difficult to analyse low-molecular-weight compounds because of the interference of matrix ion signals. Here, we report a matrix-free on-chip pulse-heating desorption/ionization (PHDI) method for a wide range of biomolecules ranging from low molecular-weight substances such as glycine (75.7 Da) to large species such as α-lactalbumin (14.2 kDa). Compared with the conventional MALDI, the matrix-free PHDI method affords high yields of singly charged ions with very less fragmentation and background using only one-pulse without light (laser). We believe that this new technique for matrix-free biomolecules analysis would overcome the limitations of the conventional MALDI.