Herein, a novel fluorescence nanosensor using intrinsic fluorescent polydopamine nanoparticles (PDA NPs) as an effective signal reporter has been constructed for the simple, rapid and sequential detection of mercury ions (Hg²⁺) and L-ascorbic acid (AA) based on a coordination effect and redox reaction. The fluorescence of the PDA NPs could be specifically quenched by Hg²⁺ through intense coordination effects between the Hg²⁺ and the groups (catechol, amine, ketone and imine) on the surface of the PDA NPs. However, when AA and Hg²⁺ coexisted in solution, the fluorescence of the PDA NPs pronouncedly recovered via the redox reaction of Hg²⁺, with it being reduced to Hg⁰ by AA. The fluorescence quenching mechanism of Hg²⁺ towards the PDA NPs and the redox reaction between Hg²⁺ and AA were also fully investigated. The nanosensor exhibited high sensitivity and desirable selectivity for Hg²⁺ and AA detection. Moreover, the strategy was successfully explored in real samples (tap water, lake water and human serum samples) with satisfactory recoveries. The developed nanosensor provides new sights and good inspiration for Hg²⁺ and AA detection under real conditions.