Background Grouper (Epinephelus spp.) is an important commercially farmed marine fish species in China and the coastal countries of Southeast Asia, and its artificial culture is highly dependent on high-quality protein sources centered on fish meal. However, the global decline in fishery resources has led to a tightening supply and rising prices of fish meal, severely constraining the sustainable development of the grouper aquaculture industry. Consequently, the development of sustainable alternative protein sources to fish meal has become a research priority in this field. Analysis This paper systematically reviews the mechanisms by which the main antinutritional factors in plant protein sources affect grouper, the appropriate substitution levels of various plant protein sources for fish meal in grouper feeds, and the negative effects of excessive substitution, including reduced growth performance, intestinal structural damage, and metabolic disorders, while also summarizing mitigation strategies using various functional additives. In addition, the effects of replacing different proportions of fish meal with various animal protein sources and single-cell protein sources on grouper are analyzed. Prospect Future research should focus on the composite formulation of multiple protein sources, the precise application of functional additives, fermentation or enzymatic pretreatment of alternative protein sources, and the construction of health assessment systems based on molecular regulatory networks, so as to promote the precision formulation and industrial application of low fish meal feeds and support the green and efficient development of the grouper aquaculture industry.