Cell signaling is a fast, dynamic, and complex process, which controls a variety of critical physiological functions. Methods to investigate such dynamic information, however, suffer from limited throughput in the single-cell level and a lack of precise fluid manipulation. Herein, we present a new strategy, termed dynamic microfluidic cytometry (DMC), for high-throughput probing of G protein-coupled receptor (GPCR) signaling in single-cell resolution (single-cell cellomics analysis) by creatively applied cyclical cell trapping, stimulating, and releasing automatically. Dose-response curves and half-maximal effective concentration (EC50) values for HeLa cells treated with adenosine triphosphate (ATP), histamine (HA), and acetylcholine chloride (ACH) were successfully obtained in the single-cell level. High-throughput single-cell dynamic signaling was further implemented by sequential or simultaneous stimulation, which revealed that different mechanisms were working in triggering intracellular calcium release. In addition, simultaneous stimulation to two different types of cells, HeLa and NIH-3T3 cells, was also successfully realized, which was crucial for online comparison of dynamic signaling of different types of cells. We believe that the proposed DMC provides a versatile means for high-throughput probing single-cell dynamic signaling, which is potentially useful in chemical biology, cell biology, and pharmacology.