In this study, the mixed strains, including Lactobacillus plantarum (STBL1), Saccharomyces cerevisiae (STBS1), and Bacillus safensis (SQVG18) were applied to the solid-state fermentation of commercial feed for Penaeus vannamei. Firstly, the optimum combination of strain inoculum, optimum fermentation conditions, and nutritional quality of the fermented feeds were investigated. It was noted that the acid-soluble protein and the content of amino acids (lysine, methionine, etc.) were remarkably improved in the feeds by solid-state fermentation with the optimum combination of STBL1: STBS1: SQVG18 = 1: 3: 3. Secondly, a six-week feeding experiment was performed using one control diet without probiotics (D0: 0 CFU g−1) and three fermented diets containing different concentrations of the optimum mixed-strain combination (D1: 107 CFU g−1; D2: 108 CFU g−1; D3: 109 CFU g−1). Shrimps fed D2, and D3 groups exhibited significantly better growth performance and activities of antioxidant enzymes and digestive enzymes than shrimp of other groups (P <.05). The fermented feeds also affected the intestinal microflora of P. vannamei at the end of the feeding trial. The diversity indices, composition, and functional prediction of microbial communities showed that shrimp fed D1 and D2 groups had a better microecological environment in intestines. Overall, the fermented diet with mixed strains of 108 CFU g−1 may be conducive to the growth and health of P. vannamei by improving growth, antioxidant capacity, digestion, and gut microflora. • The process conditions and quality evaluation in fermented feed of Penaeus vannamei were investigated. • The mixed-strains compatibility had a great influence on the nutrition in feed and their optimum ratio was explored. • In vivo experiment with the different dosage of mixed strains was carried out. • The growth performance, digestive ability, intestinal flora of P. vannamei were improved with appropriate fermented diets. [ABSTRACT FROM AUTHOR]