The effects of chitosan characteristics including the degree of deacetylation, molecular weight, particle size, pH pretreatment and immobilization time on the immobilization of nitrite-oxidizing bacteria (NOB) on biopolymeric chitosan were investigated. Nitrite removal efficiency of immobilized NOB depended on the degree of deacetylation, particle size, pH pretreatment on the surface of chitosan and immobilization time. Scanning electron microscope characterization illustrated that the number of NOB cells attached to the surface of chitosan increased with an increment of immobilization time. The optimal condition for NOB immobilization on chitosan was achieved during a 24-hr immobilization period using chitosan with the degree of deacetylation larger than 80% and various particle size ranges between 1-5 mm at pH 6.5. In general, the NOB immobilized on chitosan flakes has a high potential to remove excess nitrite from wastewater and aquaculture systems. © 2013 The Research Centre for Eco-Environmental Sciences, Chinese Academy of Sciences.