The intensification of aquaculture has resulted in frequent incidence of diseases in fish culture and which is limiting the fish production and subsequently resulting in economic losses to fish farmers. Antibiotics are being inevitably used for treating fish bacterial diseases which aggravated the problem of antibiotics residues in animal tissues, antimicrobial resistance and imbalance in the gastrointestinal microbiota of aquatic species, which has resulted in deterioration of human health and environment. Use of probiotics has proved the boon for fish culture and an important alternative method to reduce antibiotics use. The present study was carried out to evaluate the efficacy of viable probiotic bacterial combination viz., Bacillus subtillis AN11, Lactobacillus acidophilus and Saccharomyces cerevisiae on non-specific immune parameters and serum enzyme activities in Labeo rohita fingerlings. These three probiotics bacteria were added in feed @ 107 cfu g-1 of basal feed. Forty day feeding trial was conducted by distributing one hundred eighty rohu fingerlings for three treatments (T1, T2 and T3) and control in triplicates namely T1 (Bacillus subtillis AN11 + Saccharomyces cerevisiae), T2 (Lactobacillus acidophilus + Saccharomyces cerevisiae) and T3 (Bacillus subtillis AN11 + Lactobacillus acidophilus + Saccharomyces cerevisiae) and control feed without addition of probiotic bacteria. Live probiotics bacterial culture was sprayed on basal feed prior to feeding the treatment groups and two times a day feeding of fish was done. A significant increase (P < 0.05) in non-specific immune parameters such as respiratory burst assay, lysozyme activity and myeloperoxidase activity was observed in all treatment groups as compared to control while maximum was obtained in T3. Probiotic supplementation also remarkably improved serum enzyme SOD, and significantly reduced stress enzymes namely ALP and LDH activities in all treatment groups as compared to control. The total serum protein, albumin, and globulin were significantly increased (P < 0.05) in all the probiotic fed groups as compared to control. In addition to these serum bactericidal activity was also significantly increased (P < 0.05) in treatment groups which were fed with three probiotic bacterial combination (T3) than the T1 and T2 and also control. Hence, the results of present study indicate that feeding of combination probiotics to rohu fingerlings could be able to enhance the non-specific immunity in fish.