Resolving the temporal dynamics of cell signaling pathways is essential for regulating numerous downstream functions, from gene expression to cellular responses. Mapping these signaling pathways requires the exposure of cells to time-varying chemical signals; these are difficult to generate and control over a wide temporal range. Herein, we present an acoustofluidic chemical signal generator based on a sharp-edge-based micromixing strategy. The device, simply by modulating the driving signals of an acoustic transducer including the ON/OFF switching frequency, actuation time and duty cycle, is capable of generating both single-pulse and periodic chemical signals that are temporally controllable in terms of stimulation period, stimulation duration and duty cycle. We also demonstrate the device's applicability and versatility for cell signaling studies by probing the calcium (Ca2+) release dynamics of three different types of cells stimulated by ionomycin signals of different shapes. Upon short single-pulse ionomycin stimulation (∼100 ms) generated by our device, we discover that cells tend to dynamically adjust the intracellular level of Ca2+ through constantly releasing and accepting Ca2+ to the cytoplasm and from the extracellular environment, respectively. With advantages such as simple fabrication and operation, compact device design, and reliability and versatility, our device will enable decoding of the temporal characteristics of signaling dynamics for various physiological processes.