In this study, Moringa oleifera seed protein hydrolysate (MSPH) was included in the diet of Nile tilapia, Oreochromis niloticus, as a protein supplement for 90 days. Fish (n = 250, weighing 30.37 ± 0.05 g) were received diets with five inclusion levels of MSPH: 0, 33, 66, 99, and 132 g kg− 1 diet (MSPH0, MSPH33, MSPH66, MSPH99, and MSPH132), respectively, with MSPH0 serving as the control diet. Compared to MSPH0, a pronounced improvement in the final body weight (FBW), total weight gain (TWG), specific growth rate % (SGR %), feed conversion ratio (FCR), protein efficacy ratio (PER), and average daily weight gain (ADWG) was noticed in all MSPH groups, with MSPH132 showing the highest values. The improvement was by 6.41, 18.18, 34.22, and 47.05% for FBW, 9.87, 27.40, 51.39, and 70.46% for TWG, and 4.80, 15.20, 34.40, and 38.40% for FCR in the MSPH33, MSPH66, MSPH99, and MSPH132 groups, respectively. The intestinal histomorphometric and hematological indices of O. niloticus were significantly improved by MSPH levels. The total proteins and albumin were enhanced considerably by dietary MSPH (66 to 132 g kg− 1 diet), while the globulin was enhanced by MSPH (99 to 132 g kg− 1 diet). MSPH diets significantly enhanced the hepatic antioxidant enzymes and serum total antioxidant capacity and reduced malondialdehyde level. MSPH dietary inclusion did not alter the hepatorenal functions and their tissue integrity except for mild tubular adenomatous regenerative changes in the kidney of the MSPH132 group. In conclusion, the MSPH at a level up to 99 g kg− 1 diet could be used as a dietary inclusion in O. niloticus. These findings contribute to the sustainability of the Nile tilapia culture sector.