The picocyanobacteria Synechococcus and Prochlorococcus dominate oligotrophic oceans, where nitrogen (N) limitation is pervasive, and contribute substantially to marine primary production. However, their carbon biomass remains poorly constrained due to their extremely small cell size and the scarcity of direct measurements. Existing estimates largely rely on cellular carbon quota (Q_C) or carbon density derived from nutrient-replete laboratory batch cultures, which may not adequately capture physiological acclimation to nutrient limitation. Here, we established N-limited chemostat systems to investigate the responses of Q_C and cell size in Synechococcus and Prochlorococcus to varying degrees of N limitation. Synechococcus exhibited gradual declines in both cell volume and Q_C with increasing N limitation, whereas Prochlorococcus showed an initial sharp reduction followed by stabilization. This stabilization possibly suggested that Prochlorococcus had approached a physiological lower limit in cell volume and Q_C. Together, these results indicated a close coupling between cell size and Q_C in both genera under nitrogen limitation. In addition, by compiling data from this and previous studies on Synechococcus and Prochlorococcus, it was found that both genera maintained a nearly constant organic carbon density (~246 fg C μm^-3) across strains and environmental conditions, including nutrient supply, light intensity, and temperature, suggesting a conserved physiological trait. By further integrating datasets from picocyanobacteria and eukaryotic phytoplankton, we developed a universal C:vol model spanning a broad cell size spectrum (10^-1–10⁷ μm³). This model reduces estimation bias for picoplankton (< 2 μm³) while retaining accuracy for larger cells. Collectively, these findings improve estimates of phytoplankton carbon biomass and provide stronger constraints for marine biogeochemical models.