We report a nanoscale calcinated silicate film fabricated on a gold substrate for highly effective, matrix-free laser desorption ionization mass spectrometry (LDI-MS) analysis of biomolecules. The calcinated film is prepared by a layer-by-layer (LbL) deposition/calcination process wherein the thickness of the silicate layer and its surface properties are precisely controlled. The film exhibits outstanding efficiency in LDI-MS with extremely low background noise in the low-mass region, allowing for effective analysis of low mass samples and detection of large biomolecules including amino acids, peptides, and proteins. Additional advantages for the calcinated film include ease of preparation and modification, high reproducibility, low cost, and excellent reusability. Experimental parameters that influence LDI on calcinated films have been systemically investigated. Presence of citric acid in the sample significantly enhances LDI performance by facilitating protonation of the analyte and reducing fragmentation. The wetting property and surface roughness appear to be important factors that manipulate LDI performance of the analytes. This new substrate presents a marked advance in the development of matrix-free mass spectrometric methods and is uniquely suited for analysis of biomolecules over a broad mass range with high sensitivity. It may open new avenues for developing novel technology platforms upon integration with existing methods in microfluidics and optics.