A 105-day experiment was carried out to optimize the iron dose and investigate its effect on the spinach (Spinacia oleracea) and growth of pangasius (Pangasianodon hypophthalmus) in nutrient film technique (NFT) based aquaponics using aquaculture wastewater. The experimental setup consisted 15 individual and identical aquaponics units, with each unit containing a fish tank, NFT hydroponic, and a filtration system. Four different iron dosages T1 (1.0 mg/L), T2 (1.5 mg/L), T3 (2.0 mg/L), and T4 (2.5 mg/L) were assigned as treatments and compared with unsupplemented (control). The pangasius (3.13 ± 0.03 g) and spinach were stocked at 2.8 kg/m3 and 28 plants/m2, respectively in each aquaponic unit. During the experimental period, the spinach was harvested six times. During the fifth harvest (90th day), the highest yield was observed in T2 (881.74 ± 17.16 g) followed by T1 > T3 > T4 > C (563.67 ± 57.85 g). The nutrient analysis of plants revealed that the iron interacted with essential macronutrients and micronutrients and had a beneficial impact on their uptake, which in turn affected the plant growth and the yield of crops. A significant (P < 0.05) difference was found in the fish body weight among the treatments and control. The highest fish body weight was observed in T2 (16.34 ± 0.63 g) followed by T3 > T4 > T1 > C (11.72 ± 0.38 g). Higher plasma cortisol and glucose values were recorded in T4 and T3. Based on water quality parameters, spinach yield, proximate analysis, growth of fish, physiological responses, and nutrient analysis, an iron dose of 1.5 mg/L per fortnight can be optimum for pangasius-spinach nutrient film technique based aquaponics.