In this study, polyacrylamide gel (PAAG) was successfully used as a new embedding medium to provide the more effective maintenance of biological tissues during the sectioning process, enhancing the tissue imaging of metabolites via matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Herein, PAAG, agarose, gelatin, optimal cutting temperature compound (OCT), and ice media were used to embed rat liver and Atlantic salmon (Salmo salar) eyeball samples. These embedded tissues were then sectioned into thin slices and thaw-mounted on conductive microscope glass slides for MALDI-MSI detection to evaluate the embedding effects. The results showed that PAAG embedding has characteristics superior to those of commonly-used embedding media (e.g., agarose, gelatin, OCT, and ice) with the advantages of one-step operation without heating, a better performance of morphology maintenance, the absence of PAAG polymer-ion-related interference below m/z 2000, and the more efficient in situ ionization of metabolites, providing a significant enhancement of both the numbers and intensities of the metabolite ion signals. Our study demonstrates the potential of PAAG embedding as a standard practice for metabolite MALDI tissue imaging, which will lead to an expanded application scope of MALDI-MSI.