While the formation of intracellular amorphous calcium carbonate (ACC) by living organisms is widespread, its detection in prokaryotes remains difficult owing to its susceptibility to transform or dissolve upon sample preparation. Because of these challenges, a large number of ACC-forming prokaryotes may have been undetected and their abundance in the natural environment is possibly underestimated. This study identifies diagnostic spectral markers of ACC-forming prokaryotes that facilitate their detection in the environment. Accordingly, ACC formed by cyanobacteria was characterized using Fourier transform infrared (FTIR) spectroscopy in near-IR, mid-IR, and far-IR spectral regions. Two characteristic FTIR vibrations of ACC, at ∼ 860 cm−1 and ∼ 306 cm−1, were identified as reliable spectral probes to rapidly detect prokaryotic ACC. Using these spectral probes, several Microcystis strains whose ACC-forming capability was unknown, were tested. Four out of eight Microcystis strains were identified as possessing ACC-forming capability and these findings were confirmed by scanning electron microscopy (SEM) observations. Overall, our findings provide a systematic characterization of prokaryotic ACC that facilitate rapid detection of ACC forming prokaryotes in the environment, a prerequisite to shed light on the role of ACC-forming prokaryotes in the geochemical cycle of Ca in the environment.