Graphene, graphene oxide (GO), and multi-walled carbon nanotube (MWCNT) have been actively explored as matrix for laser desorption/ionization mass spectrometry (LDI-MS) in place of conventional organic matrix. Recently, the GO/MWCNT double layer films on a solid substrate showed excellent applicability in small molecule analysis, enzyme activity assay and tissue imaging. In the present study, LDI processes of small molecules on multilayers of alternating MWCNT and GO hybrid films were thoroughly investigated. We found that the LDI efficiency of small molecules was affected by thickness, assembly sequence and surface roughness of the hybrid films that were controlled by varying the number of layer-by-layer (LBL) assembly cycles. The study would provide useful basic information to develop an efficient LDI-MS platform based on carbon nanomaterials.