A 60-day experimental trial was conducted to evaluate the effect of different stocking densities, probiotic supplementation, and a biofloc system on the growth and physiological responses of Puntius sophore in laboratory conditions. P. sophore (8.64 ± 1.24 g) was obtained from the Brahmaputra River, Mymensingh, and immediately transferred to a flow-through water system. In experiment 1, fish were subjected to three treatments (20 fish per 400 L as LD, 25 fish per 400 L as MD, and 30 fish per 400 L as HD), and similarly, in experiment 2, three different types of diets were provided (control diet (D1), biofloc (D2), and a probiotic-containing diet (D3)). Three replications were used in the completely randomized experimental design. Growth parameters, viz. TLG, BWG, FCR, FCE, SGR, RGR, and DGR, were significantly influenced by stocking density and probiotics (p < 0.05). The highest growth rate was observed in LD and in D3. The lowest FCR was also observed in LD and in D3, while highest was in HD (30 fish per 400 L) and in D2. Though stocking density does not impact the blood profile, with the exception of WBC, in the case of feeding regime, WBC, RBC, MCV, MCH, and PLT levels differed significantly (p < 0.05); however, all haematological measures were within the normal range in both experiments, and the fish’s physiological conditions were better in LD and feeding with probiotic supplementation (D3). The results obtained from this study suggested that the welfare of P. sophore is adversely affected by high stocking density and increased growth and physiological conditions when cultured with LD and supplemented with probiotics.