Photosynthesis is the most important bioenergy conversion process on Earth. Capturing instantaneous changes in in situ photosynthesis in open ocean ecosystems remains a major challenge. In this study, fast repetition rate fluorometry (FRRF), which can obtain nondestructive, real-time and in situ estimates of photosynthetic parameters, was used for the first time to continuously observe the spatial variation in in situ photosynthetic parameters in the eastern Indian Ocean (EIO). We further formulated new insights regarding abiotic and biotic factors of potential importance in determining photosynthetic performance. First, we found that the distributions of micro/nano- and picophytoplankton were opposite under the control of nutrient concentrations. Micro/nanophytoplankton had higher cell abundances in the nearshore and upwelling regions, while picophytoplankton had higher abundances in the open ocean, and Prochlorococcus was the dominant group. Second, based on the FRRF technology, we obtained the high-precision and high-density vertical profile map of photosynthetic parameters in the euphotic layer. It was observed that values of the maximum photochemical efficiency (Fv/Fm; 0.14–0.55, unitless) and the functional absorption cross-section of PSII (σPSII; 1.71–4.90 nm2 RCII−1) increased with increasing depth, while high values of the photosynthetic electron transfer rates (ETRRCII; 0.0019–17.0292 mol e− mol RCII−1 s−1) and the nonphotochemical quenching (NPQNSV; 0.35–7.26, unitless) occurred in the shallow 50 m layer, and the values decreased as the depth increased. Finally, we discussed limiting factors that regulated the distribution of photosynthetic parameters and concluded that optical properties varied significantly with changes in the ocean physico-chemical parameters and taxonomic composition of phytoplankton assemblages in the EIO. Picophytoplankton (especially cyanobacteria), rather than the micro/nanophytoplankton community, was the dominant factor influencing photosynthesis. Among abiotic factors, photosynthetically active radiation (PAR) was the proximal limiting factor affecting photosynthetic efficiency, followed by temperature and dissolved inorganic nitrogen (DIN). Consequently, phytoplankton photosynthetic parameters exhibited great variability, allowing rapid responses to environmental condition changes. In this study, we established the basis for detecting future changes in primary production in this oligotrophic area.