A defective three dimensionally ordered macro-mesoporous TiO2-X (3DOMM-TiO2-X) was successfully prepared through dual-template method and applied as non-enzymatic electrochemical glucose sensor. The introduced oxygen vacancies and Ti3+ defects through defect engineering, as well as the macro-mesoporous structure induced by dual template, synergistically improve the performance of glucose determination. The Nafion/3DOMM-TiO2-X/FTO electrode shows better current response to glucose than commercial P25 and 3DOM-TiO2. A two-stage wide detection linear range of 1 mu M similar to 2.32 mM and 2.32 mM similar to 14.72 mM was observed, with the sensitivity of 14.11 mu A mM(-1) cm(-2) and 3.44 mu A mM(-1) cm(-2), detection limits of 0.15 mu M and 0.63 mu M (S/N = 3), respectively. The electrode also exhibits superior reproducibility, stability, and selectivity performance. Furthermore, a reactive oxygen species oxidation mechanism was proposed and investigated. The Nafion/3DOMM-TiO2-X/FTO electrode also exhibits the potential as a photoelectrochemical electrode. Our results indicate the feasibility of the strategies to enhance the sensing ability by improving electronic conductivity through constructing 3DOMM porous structure and defect engineering.