A novel technique, termed matrix coating assisted by an electric field (MCAEF), for enhancing tissue imaging by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was developed in this study. In this technique a static and uniform electric field is applied to sliced tissue sections during matrix spray-coating, resulting in the enrichment of positively or negatively chargeable analytes in the MALDI matrix layer. Experimental results show that MCAEF not only increased the sensitivity of lipid and protein detection across the board in the subsequent MALDI-MS analyses, but also resulted in successful imaging of a larger number of analytes. MALDI imaging enhancement with MCAEF was observed for various tissues (rat liver, rat brain, and porcine adrenal gland) and with different MALDI matrices (e.g., quercetin, 2-mercaptobenzothiazole, dithranol, 9-aminoacridine, and sinapinic acid) and the sensitivity increases were independent of the solvent compositions and pH values of the matrix solutions. Taking rat brain as an example, MCAEF led to the on-tissue detection and imaging of 648 identified lipids by combining positive and negative ion detection by MALDI-Fourier transform ion cyclotron resonance MS and with quercetin as the matrix, as compared to only 344 lipids without MCAEF. For protein imaging, up to 232 protein signals were successfully detected in rat brain tissue sections by MALDI-time-of-flight MS within a mass range of 3500 to 37 000 Da, as compared to 119 without MCAEF. MCAEF also enabled the detection of higher molecular-weight proteins. These results demonstrate the advantages of MCAEF for overall performance improvements in MALDI imaging and we believe that this technique has the potential to become a standard practice for MALDI tissue imaging.