The objective of the current study was to assess the impact of dietary phytase supplementation on Labeo rohita fingerlings and to examine the effects on growth, nutrient digestibility and chemical characteristics of diets containing rice protein concentrate (RPC) as a major protein source. Six experimental diets were made, i.e., a positive control (fishmeal-based diet with no phytase), FM0; a negative control (RPC-based diet with no phytase), RPC0; and four supplemental phytase levels (250, 500, 1000, and 2000 FTU/kg). Fingerlings with an average weight of 9.42 ± 0.02 grams (mean ± SD) were randomly distributed into six experimental groups of three replicates, each containing 25 fish per tank (75 liters of water), provided with experimental diets at a rate equivalent to 5% of their body weight for 90 days, and uneaten feed was collected after 2 hours to determine feed consumption. The feces were collected before feeding to estimate digestibility. Phytase in combination with the RPC-based diet significantly (p < 0.05) enhanced phytate phosphorus in vitro hydrolysis; growth performance; nutrient (crude protein, crude fat, moisture and gross energy) and mineral (P, Ca, Mg, Na, K, Zn, Mn and Cu) digestibility; digestive enzyme (protease, lipase and amylase) activity; and mineral deposition up to 1000 FTU/kg phytase. However, the hepatosomatic and viscerosomatic indices and carcass composition were not influenced (p > 0.05) by phytase supplementation. Increasing phytase supplementation in the RPC-based diets led to a significant (p < 0.05) decrease in the serum biochemical parameters (alkaline phosphatase activity, aspartate aminotransferase, alanine aminotransferase), which resulted in improved liver health. In conclusion, phytase-supplemented RPC-based diets improved the growth, mineral/nutrient digestibility, digestive enzymes, serum biochemistry, and mineral deposition of L. rohita fingerlings up to 1000 FTU/kg. Broken line regression analysis revealed that the optimum phytase concentration in the RPC-based diet for L. rohita was 874.19 FTU/kg.