Fluorescence microscopic methods are critical for spatial profiling of multiple biological targets in cells and tissues to study cell and tissue functions, but their multiplexity was limited to 3~5 targets under a conventional setup using different fluorescent channels because of spectra overlap. Here, we introduce a simple, rapid, multiplexed fluorescence imaging method in cells and tissues, termed DNA based plasmonic heating activated signal exchange reaction (PHASER). PHASER uses infrared light-induced plasmonic heating of gold bipyramid nanoparticles to sequentially activate thermodynamically calibrated DNA thermal probes in situ for rapid and multiplexed fluorescent imaging of biological targets. We showed that the signal exchange per round between biological targets in PHASER can be completed within 30 seconds, and that 5 irradiation pulses of photothermal heating can activate DNA thermal probes with 5 different signal temperatures in cells and tissues. To demonstrate its practical use, we applied PHASER to profile the subcellular spatial organization of different organelles in cultured cells and resolved different protein spatial expression profiles in mouse brain tissue with dimensions of millimeters in a single fluorophore channel. PHASER is expected to have broad biotechnical applications with multiplexed fluorescence imaging for a wide variety of biological targets across diverse samples.