Evaluation of various immobilization methods and bioreactors for sulfide oxidation using Thiobacillus sp. was studied. Ca-alginate, K-carrageenan and agar gel matrices (entrapment) and polyurethane foam and granular activated carbon (adsorption) efficacy was tested for the sulfide oxidation and biomass leakage using immobilized Thiobacillus sp. Maximum sulfide oxidation of 96% was achieved with alginate matrix followed by K-carrageenan (88%). Different parameters viz. alginate concentration (1%, 2%, 3%, 4% and 5%), CaCl2 concentration (1%, 2%, 3%, 4% and 5%), bead diameter (1, 2, 3, 4 and 5 mm), and curing time (1, 3, 6, 12 and 18 h) were studied for optimal immobilization conditions. Repeated batch experiments were carried out to test reusability of Ca-alginate immobilized beads for sulfide oxidation in stirred tank reactor and fluidized bed reactor (FBR) at different sulfide concentrations. The results proved to be promising for sulfide oxidation using Ca-alginate gel matrix immobilized Thiobacillus sp. for better sulfide oxidation with less biomass leakage. Biological sulfide oxidation is gaining more importance because of its simple operation. Present investigations will help in successful design and operation of pilot and industrial level FBR for sulfide oxidation.