All-inorganic CsPbBr3 perovskite quantum dots (QDs) are widely studied owing to their excellent optoelectronic properties; however, they are usually hydrophobic and unstable in water and thus their biomedical applications are seriously limited. In this study, stable and hydrophilic CsPbBr3 QDs functionalized with carboxyl groups (CsPbBr3-COOH QDs) were prepared in one-pot with the aid of new ligands amino-poly(ethylene glycol)-carboxyl and perfluorooctyltriethoxylsilane. The aqueous solution of CsPbBr3-COOH QDs maintained the initial fluorescence intensity after 8 days of storage; the free carboxyl groups on the surface of CsPbBr3-COOH QDs were covalently conjugated with amino-terminal DNA to construct CsPbBr3 QDs-DNA probes for subsequent application. Then, a biosensing platform utilizing fluorescence resonance energy transfer between hydrophilic CsPbBr3 QDs-DNA and MoS2 nanosheets was developed for the sensitive and selective detection of the Mycobacterium tuberculosis DNA with a low limit of detection of 51.9 pM and the identification of drug-resistant clinical strains. This study advances the preparation of hydrophilic carboxyl-functionalized CsPbBr3 QDs with enhanced stability and extends their application in biomolecule detection.