The search for suitable fish meal replacements in aqua-diets is a salient agenda in the constant effort of making aquaculture practices more sustainable. In this study, we tested four customised diets composed by systematic inclusion of pre-selected fish meal substitutes, lupin kernel meal, BSF meal, TH and PBM on growth, metabolism, cytokine profile, gut morphology and microbiota of juvenile Lates calcarifer. Five isoproteic and isoenergetic diets were prepared viz. FM100 as a control (without fish meal substitute), while FM75, FM50, FM25 and FM0 indicates replacement of fish meal (FM) at 25%, 50%, 75%, and 100%, respectively by a mixture of four different pre-selected non-fish meal (NFM) ingredients. Fish fed FM100, FM75, FM50, FM25 exhibited consistent growth and haematological response, while the fish fed no fishmeal (FM0) showed significant decline in final body weight (FBW) and specific growth rate (SGR). The poor growth performance was correlated with a decrease in villous width, microvilli height and goblet cells density. A significant shift in abundance profile of Psychrobacter in the gut microbial profile of fish fed FM50 was noticed compared to fish fed FM100. The results of qRT-PCR showed up-regulated expression of innate immune responsive genes in the FM50 group. The adverse impacts on growth performance and gut health of fish fed FM0 suggest that the complete substitution of fishmeal is not advisable and the inclusion range of these alternatives should be decided for a species only after examining their effect on maximal physiological performance.