The invention discloses a DntR mutant and an application thereof to detection of o-dihydroxybenzene. Directional modification of DntR regulatory gene is carried out in order to construct a mutant library of large capacity, a biological effector with green fluorescent protein as a reporter gene is established, a high throughput screening method sorted by a flow cytometer is combined, and a o-dihydroxybenzene biological sensing molecule is obtained. The biological sensing molecule can be applied to detection of discharge standard of industrial sewage treatment and residual quantity of pollutants in environment, and also can be applied to high throughput screening of AP degradation key enzyme gene directional modification.