We demonstrate continuous-wave laser-based ambient mass spectrometry imaging with the use of a gold-nanoparticle layer substrate. When a fresh tissue slice is placed on a gold-nanoparticle layer substrate and irradiated with a 532-nm continuous-wave laser, the transmission-mode laser configuration provides precise desorption performance to facilitate mass spectrometry imaging. The subsequent ionization process with non-thermal atmospheric pressure plasma jets generates sufficient amounts of molecular ions. By using this method, micrometer-spatial-resolution mass spectrometry imaging of humid tissues can be obtained. The gold-nanoparticle layer substrates can be prepared and stored in advance of the experiment, resulting in simplified specimen preparation and an advantage in faster preparing of fresh tissue specimen for analysis.