Calcium ions (Ca2+) are key players in intracellular signaling as second messengers and play a pivotal role in various physiological processes. In this study, near-infrared water-soluble AgInS2 quantum dots (AIS QDs) for Ca2+ detection were synthesized by a one-step hydrothermal method. The fluorescence quantum yield (PL QY) of the quantum dots was as high as 23.99 %. With low cytotoxicity and good fluorescence properties, as well as short reaction time, the ternary AIS QDs have excellent synthesis efficiency and quantum yield, which are advantageous for Ca2+ detection and bioimaging applications. The fluorescence quenching of the quantum dots showed a clear linear relationship with calcium ion concentration in the range of 0–250 μM (detection limit: 0.65 μM). Confocal imaging experiments demonstrate the excellent biofluorescence imaging capability of AIS QDs. By tuning the Ag/In molar ratio, AIS QDs can achieve fluorescence emission in the near-infrared wavelength band (620–700 nm), and the near-infrared fluorescence imaging has deeper tissue penetration, less tissue absorption and photodamage, and lower interference of spontaneous fluorescence, which further expands the potential of QDs for bioimaging applications.