High-performance determination of pyrophosphate ions (PPi) and pH is an important goal in biological systems. In this work, Cu-doped carbon dots (Cu-CDs) were synthesized rapidly and simply via a one-pot hydrothermal method. The as-obtained Cu-CDs, with an average size of 2.55 nm, exhibit an excitation-independent fluorescence emission and possess desirable functional groups of carboxyl and amine, which can be served as fluorescence nanoprobes for detection of PPi based on surface passivation. Under the optimal condition, the linear range for detection of PPi was 0.05-20 µM, and the corresponding limit of detection (LOD) was 0.013 µM, indicative of a promising assay for the PPi. Moreover, the fluorescent intensity of the Cu-CDs is linear against pH value from 6 to 8.7 in buffer solution, suggesting the feasibility as a pH sensor. The synthesized Cu-CDs coated fluorescent paper indeed can monitor pH in urine with satisfaction by naked eyes through ultraviolet irradiation. The successful detection of PPi and the visual detection of pH value suggest a highly promising application of Cu-CDs in the field of biosensing.