Abstract The cellular size and biomass of picophytoplankton were studied by flow cytometer during spring monsoon (March–May of 2015) in equatorial eastern Indian Ocean. We established an empirical relationship between forward scatter and cellular size to address the size and biomass of picophytoplankton. Results indicated that mean cell diameter of Prochlorococcus (0.60 μm) was the smallest, and then followed by Synechococcus (0.98 μm) and picoeukaryotic phytoplankton (1.05 μm). Thereafter, the biomass converted by abundance reached 0.64 μg·C·L −1 for Prochlorococcus , 0.34 μg·C·L −1 for Synechococcus , and 0.20 μg·C·L −1 for picoeukaryotic phytoplankton. Additionally, the distinct biomass contribution of picophytoplankton appeared to be affected by abundance, but not changes in cellular size. Vertically, the cellular sizes of picophytoplankton were remarkably small in upper waters, which was predominantly controlled by the nutrient availability. In contrast, they were larger in deeper waters, which was primarily attributed to the combined effects of low temperature and reduced light availability. Spatially, under the influence of high nutrient concentration induced by the different circulations and coastal upwelling, slightly high carbon biomass of picophytoplankton was observed around the coastal zones of Sri Lanka island and Sumatra, as well as the southern Bay of Bengal.