The impact of ocean acidification on the physiology and ecology of microalgae is a focal topic in global change research. However, direct quantitative evidence at the single-cell level remains scarce regarding how intracellular inorganic carbon systems of different functional groups of microalgae respond differentially to acidification stress. In this study, the diatom Ditylum brightwellii and the dinoflagellate Prorocentrum dentatum were selected as representative species and an acidification treatment group (pCO₂ = 1000 ppm) and a control group (pCO₂=400 ppm) were set. Concentrations of the carbonate system parameters—including dissolved inorganic carbon (DIC), total alkalinity (TA), partial pressure of CO₂ (pCO₂), and pH—within individual cells of both microalgae over a complete growth cycle were determined using an ultrasonic disruption method to obtain the difference between pre-disruption and post-disruption operations. Concurrently, the dynamics of chlorophyll content and nutrient concentrations were monitored. The results indicated that generally the mean intracellular DIC concentrations were 1.01×10⁷ μmol/L for Ditylum brightwellii and 1.11×10⁵ μmol/L for Prorocentrum dentatum, respectively. Under acidification, intracellular DIC concentrations of Ditylum brightwellii. exhibited an overall declining trend over the growth cycle, accompanied by an increase in TOC and a decrease in the DIC/TOC ratio, suggesting that acidification promoted the conversion of inorganic carbon to organic carbon. In contrast, Prorocentrum dentatum. displayed a markedly greater magnitude of change in intracellular DIC concentrations under the same acidification stress, likely because its relatively thin cell wall cannot effectively impede H⁺ influx. The ultrasonic disruption-difference method established in this study provides a novel technical approach for the multi-parameter quantification at the single-cell level in microalgae. The findings unveil the distinctions in intracellular inorganic carbon allocation and regulatory strategies between diatoms and dinoflagellates, thereby offering reference data and a theoretical basis for predicting the succession trends of marine microalgal functional groups under future ocean acidification scenarios.