Biological nitrogen fixation is much more important process in nitrogen cycle of the oceans than what was previously thought of. Nitrogen fixation may have an influence on capacity of the oceans to sequester carbon and the interactions between photosynthetic production of organic matter and nitrogen nutrient dynamics in the sea. A greater diversity of marine nitrogen fixation cyanobacteria has also been unknown and their quantitative significance remains to be determined. It is necessary to further study diversity and abundance of N₂ fixing cyanobacteria in order to resolve the issue of N₂ fixation in the ocean. A variety of approaches can reveal the the N₂ fixing ability of cyanobacteria, such as stable isotope tracer techniques to measure N₂ fixation rates; natural abundance stable isotope studies that indicate the contribution of N₂ fixation to organic matter; visual examination of microbial assemblages by microscopic approaches and genetic studies targeted at the genes for N₂ fixation. The application of classical microbiological, physiological and molecular techniques has drawn an increasingly complex picture of the composition and roles of marine cyanobacteria populations. Present study suggests that N₂ fixation could indeed be more widespread and important than those was previously assumed, and indicates that they might make a substantial contribution to the open ocean nitrogen budget. Significance of nitrogen fixation was discussed based on its recent results and protocols with specific emphasizes on its influencing factors, biochemical and molecular study. Then, scientific questions were launched for further research on marine nitrogen fixation, such as N₂ fixing picoplankton, the extent and diversity of N₂ fixing symbionts and genetic diversity of symbiotic cyanobacteria, aiming at offering basics for nitrogen fixation study.