The graphene sheets prepared by chemical reduction of graphene oxide were utilised as the precursor and the graphene film can be obtained by vacuum filtration of the as-synthesised graphene suspension. A pulsed laser engineering approach is developed to prepare the functional graphene film with graphitic nanospheres homogeneously decorated on the surface. As a result of laser engineering process, the as-made graphene film enjoys micro- and nano-scale surface roughness with better stability. Meanwhile, the surface hydrophobicity is enhanced and electric conductivity is improved after laser treatment. The structural parameters of the functional graphene films may contribute to advanced applications in mass spectrometry detection, where laser desorption/ionization mass spectrometry (LDI MS) detection of analytes was performed on the engineered graphene films without using conventional organic matrix. The graphene film is (1) stable with minimum matrix interference; (2) able to provide sufficient surface area and desirable interaction with analyte molecules; (3) electric conductive with high electron mobility and efficient in laser absorbance for desorption/ionization. Consequently, the detection of limit can reach femtomolar level towards diverse molecules in MS analysis.