The acidic catalysts play pivotal roles in the dehydration of hexose into 5-hydroxymethylfurfural (HMF). However, these acidic catalysts were usually exogenously added or self-made, which increase the cost of preparation of HMF. Herein, a novel route to prepare HMF from high fructose corn syrup (HFCS-55) using gluconic acid in situ produced by bio-oxidation as a catalyst has been investigated. In the process of bio-oxidation of HFCS-55, the introducing of E-polylysine (EPL) can effectively improve the acid tolerance of the coimmobilized glucose oxidase and catalase. The immobilized enzymes can keep activity in the existence of a large amount of gluconic acid. Through the chelation of gluconic acid and calcium ions, the degree of gluconic acid ionization was raised and enough protons were released to convert fructose to HMF without the addition of acid. Using 2-methyltetrahydrofuran (2-MeTHF) as an extractor and adding 2 wt % CaCl2, the HMF's yield reached 85% from fructose-gluconic acid mixture solution at 150 degrees C for 10 min with 200 W microwave irritations. Furthermore, we also used nuclear magnetic titration to investigate the complexation mechanism of the calcium ion and gluconic acid.