The diazotroph-derived nitrogen (DDN) is an important source of bioavailable nitrogen in the marine ecosystem. Release and transfer of DDN derived by Trichodesmium, play key roles in regulating both the primary production and export production in the oligotrophic ocean. However, previous studies have shown great uncertainty in direct release rate of Trichodesmium derived nitrogen, which probably induced by the different detection method they used, or the indirect release caused by exogenous processes. As a result, the direct release process of DDN and its mechanisms are poorly understood. Furthermore, the transfer of DDN derived by Trichodesmium to major phytoplankton species in the oligotrophic ocean, remain to be revealed. Although studies have reported enhanced direct DDN release by Trichodesmium under both high and low light condition, the mechanism behind remains to be elucidated. Therefore, in this study, we establish a stable and reproducible co-culture system of Trichodesmium and Synechococcus in the laboratory to examine the DDN release rate of Trichodesmium and its transfer to Synechococcus. Stable isotopic tracing results show that most of DDN (>90%) was utilization by Trichodesmium itself during exponential phase; the release and transfer of DDN increased with increased light. We offer a method to explore the factor control DDN release and transfer, and provide an insight into the importance of Trichodesmium to both primary production and export production in the oligotrophic ocean.