Abstract: Biological nitrogen fixation (BNF) is the only biological process that can convert the “free” atmospheric nitrogen into chemical nitrogen for the utilization of plant and microorganism. Leguminous crops are widely known about their high efficiency in symbiotic nitrogen fixation, and have been used in agriculture as a supplement of nitrogen for other crops. Recent studies found that gramineous crops also had powerful associative nitrogen fixation capacity. In particular, large numbers of associative nitrogen-fixing bacteria have been identified in root, rhizosphere, and the above ground compartments of plants, like stem vascular bundle and phyllosphere, suggesting that nitrogen-fixing microorganisms in gramineous crops may have created a new pathway for “efficient nitrogen fixation in vivo” to avoid complex soil environments. This article presents an overview of the recent advancements in the associative nitrogen fixation compartments, regulatory pathways, and microbial community construction of gramineous crops, such as maize, wheat, rice, and sugarcane. This review focuses on the potential interactions between nitrogen-fixing bacteria and other functional bacteria, fungi, and viruses, beyond their interactions with host plants. Based on the promising application prospects of multifunctional synthetic microbial community in the fields of plant nutrition and growth promotion, this paper outlines the current research frontiers and challenges in associative nitrogen fixation of gramineous crops. The ultimate aim is to integrate the “top-down” and “bottom-up” strategies to identify key functional groups, combine genome-scale metabolic models, and construct stable, functionally diverse, and effective synthetic microbial community, and to provide robust technical support for the widespread application of BNF in agricultural production.