The development of on-tissue chemical derivatization methods for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) of small endogenous metabolites in tissues has attracted great attention for their advantages in improving detection sensitivity and ionization efficiency of poorly ionized and low abundant metabolites. Herein, a laser-assisted tissue transfer (LATT) technique was developed to enhance on-tissue derivatization of small molecules. Using a focused blue laser, a thin-layer tissue film (∼1 μm) was transferred to an acceptor slide from a 6 μm dry tissue section preliminarily coated with derivatization and matrix reagents. The acceptor slide with its ablated constituents was then imaged by MALDI MS. On-tissue chemical derivatization with amino-specific derivatization reagent 4-hydroxy-3-methoxycinnamaldehyde (CA) was carried out on LATT system. 20-235 folds increase in signal intensity for CA derivatized metabolites such as amino acids, neurotransmitters, and dipeptides were observed from rat brain tissues in comparison with conventional incubation-based derivatization. This technique was further extended to derivatize steroids with Girard reagent T (GirT). The remarkable derivatization efficiency can mainly be attributed to the minimization of ion suppression effects due to the reduced thickness of tissue section and endogenous components. Additionally, shorter derivatization time with no obvious metabolite delocalization was achieved using LATT method. These results demonstrate the advantages of LATT in the enhancement of on-tissue derivatization for the more specific and sensitive imaging of small metabolites in tissues with MALDI MS.