Glucose isomerase is capable for catalyzing D-glucose to form D-fructose. The obtained high fructose corn syrup (HFCS) is widely served as sweetener in various food fields. This study addressed the immobilization conditions optimization of recombinant Escherichia coli BL21 (DE3)/pET28b/ToGI using tetrakis(hydroxylmethyl)phosphonium as crosslinker. It was confirmed that the optimum particle diameter of diatomite carrier was 36.8 μm, the optimum molecular weight of polyethylene imine was 70000, and the optimum forming precursor of crosslinker was tetrakis(hydroxylmethyl)phosphonium sulfate. Furthermore, the optimum immobilized process was determined at the condition of 0.3 g carrier, 5 mmol/L Mn2+, 0.12% (v/v) polyethylene imine flocculation for 1.0 h, and 1.5% (v/v) tetrakis(hydroxylmethyl)phosphonium sulfate crosslinking for 1.5 h. The activity of the resulting immobilized cells reached 138.4 U/g. After 10 batches of bioconversion at 85 ℃ and 1 100 mmol/L D-glucose, the biocatalyst still exhibited at least 90.3% of initial activity and all the yield of D-fructose was above 51.7%. Successive production of HFCS with higher D-fructose using this craft has the advantage of simplifying the subsequent isolation and extraction step, and it has an important reference value for industrial application in HFCS production using food safety expression system.