Dysfunction of mitochondria is closely related to neurodegenerative diseases, heart diseases, cancers, and so on. Because both proton and oxygen participate in vital biochemical reactions occurring in mitochondria such as adenosine triphosphate (ATP) generation, measuring proton and oxygen concentrations in mitochondria is therefore crucial for monitoring mitochondria activities and understanding cellular behavior. For this purpose, we developed a ratiometric fluorescent nanosensor for simultaneously sensing and imaging O2 and pH in mitochondria. The steps are as follows: (1) Styrene was copolymerized with 2-aminoethyl methacrylate hydrochloride to produce amino-functionalized polymer nanoparticles. (2) The reference dye rhodamine B isothiocyanate (RBITC) and oxygen-sensitive dye platinum(II) octaethylporphyrin (PtOEP) were encapsulated into a polymer sphere during polymerization, while the pH indicator fluorescein isothiocyanate (FITC) and mitochondrial-targeting molecule (3-carboxypropyl)triphenylphosphonium bromide (TPP) were further modified on the surface of the nanoparticles. The developed nanosensor shows a narrow distribution of particle size, high sensitivity toward O2 and pH, excellent stability, and low cytotoxicity. These remarkable features of the dual nanosensor render them capable of real-time sensing and imaging of O2 and pH in mitochondria with high spatial resolution. Applying the mitochondrial-targeted dual nanosensor in HeLa cells, we quantitatively measured and imaged mitochondrial proton and oxygen concentration variations after carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment.