The cellular response to the complex extracellular microenvironment is highly dynamic in time and type of extracellular matrix.Accurately reconstructing this process and analyzing the changes in recep-tor conformation on the cell membrane surface and intracellular or intercellular signaling has been a major challenge in analytical chemistry and biophysical methodology.In this paper,a time-coded multi-concentration microfluidic chemical waveform generator was developed for the dynamic signaling prob-ing with single-cell array of high temporal resolution,high throughput,and multi-concentration combina-tion stimulation.Based on innovative microchannel structure,sophisticated external control methods and multiplexing technology,the system not only allowed for temporally sequential permutations of the four concentrations of stimuli(time code),but also generated pulsed and continuous waveforms at different frequencies in a highly controllable manner.Furthermore,the single-cell trap array was set up to effi-ciently capture cells in suspension,dramatically increasing throughput and reducing experiment prepara-tion time.The maximum frequency of the platform was 1 Hz,and one cell could be stimulated at multi-ple frequencies.To show the ability of the system to investigate rapid biochemical events in high through-put,pulse stimulation and continuous stimulation of different frequencies and different time codes,com-bined with four concentrations of histamine(HA),were generated for probing G protein-coupled receptor(GPCR)signaling in HeLa cells.Then,statistical analysis was performed for the mean peak height and mean peak area of the cellular response.We believe that the time-coded multi-concentration microflu-idic chemical waveform generator will provide a novel strategy for analytical chemistry,biophysics,cell signaling,and individualized medicine applications.