Two novel Hg2+ fluorescent probes W-5 and W-6 have successfully been designed and synthesized based on benzothiazole derivatives as fluorophores and thiocarbonate moiety as recognition group. The structures of W-5 and W-6 were confirmed by NMR, HRMS and X-ray crystallography analysis. The probes W-5 and W-6 showed the ratio (F525/F405, large emission shift 120 nm) and “turn-on” fluorescence signals, respectively. Their advantages are excellent selectivity and anti-interference. Moreover, the probes W-5 and W-6 can provide excellent methods for the determination of Hg2+ in BSA and calf serums.