This study introduces a ratiometric fluorescent sensing method for inorganic pyrophosphate (PPi) using sp³-functionalized (6,5) single-walled carbon nanotubes (SWNTs). The SWNTs are functionalized with sp³ defects that exhibit red-shifted defect emission in the near-infrared (NIR-II) range (1000–1700 nm). The sensing mechanism relies on the immobilization of Cu²⁺ ions on the SWNT surface via coordination to covalently attached aryl alkyne groups and a triazole complex. The presence of Cu²⁺ ions causes fluorescence quenching via photoinduced electron transfer, which is reversed by copper-complexing analytes such as PPi. The differences in the fluorescence response of sp³-defect to pristine nanotube emission enable reproducible ratiometric measurements over a wide concentration range. Biocompatible, phospholipid-polyethylene glycol-coated SWNTs with sp³ defects are used for detecting PPi in cell lysate and monitoring DNA synthesis in polymerase chain reaction (PCR). This robust ratiometric and NIR-II luminescent probe for PPi may serve as a basis for the rational design of nanotube-based biosensors. SWNTs are promising platforms for spectroscopic sensing in the NIR-II window due to their sensitivity to their environment. They can be seen as rolled-up graphene sheets with different roll-up angles and diameters, leading to different nanotube species, i.e., chiralities. Each species of SWNTs has characteristic optical properties and exhibits narrow photoluminescence (PL) peaks within the NIR-II window. In combination with their high photostability and biocompatibility, SWNTs are excellent materials for biosensor development. Various SWNT-based biosensors have been developed that are sensitive to, for example, bacterial motifs, reactive oxygen species, metal ions, proteins, and neurotransmitters. The intentional introduction of sp³ defects by covalent functionalization has been shown to enhance the fluorescence properties of SWNTs and increase their PL quantum yield. At low densities, sp³ defects lead to new and bright emission bands (typically labeled as E₁₁*) that are red-shifted from the native excitonic E₁₁ emission. They provide additional fluorescence signals at different wavelengths and with different responses to changes in the nanotube environment and to analytes. Thus, they enable multimodal and ratiometric detection schemes. For the easily sorted and purified species of (6,5) SWNTs (diameter 0.76 nm), the E₁₁ emission occurs at ≈990 nm and the E₁₁ emission at ≈1140 nm in aqueous dispersion. Very recently, such sp³ defects were successfully used by Kim et al. as fluorescent probes to detect ovarian cancer and by Spreinat et al.This study introduces a ratiometric fluorescent sensing method for inorganic pyrophosphate (PPi) using sp³-functionalized (6,5) single-walled carbon nanotubes (SWNTs). The SWNTs are functionalized with sp³ defects that exhibit red-shifted defect emission in the near-infrared (NIR-II) range (1000–1700 nm). The sensing mechanism relies on the immobilization of Cu²⁺ ions on the SWNT surface via coordination to covalently attached aryl alkyne groups and a triazole complex. The presence of Cu²⁺ ions causes fluorescence quenching via photoinduced electron transfer, which is reversed by copper-complexing analytes such as PPi. The differences in the fluorescence response of sp³-defect to pristine nanotube emission enable reproducible ratiometric measurements over a wide concentration range. Biocompatible, phospholipid-polyethylene glycol-coated SWNTs with sp³ defects are used for detecting PPi in cell lysate and monitoring DNA synthesis in polymerase chain reaction (PCR). This robust ratiometric and NIR-II luminescent probe for PPi may serve as a basis for the rational design of nanotube-based biosensors. SWNTs are promising platforms for spectroscopic sensing in the NIR-II window due to their sensitivity to their environment. They can be seen as rolled-up graphene sheets with different roll-up angles and diameters, leading to different nanotube species, i.e., chiralities. Each species of SWNTs has characteristic optical properties and exhibits narrow photoluminescence (PL) peaks within the NIR-II window. In combination with their high photostability and biocompatibility, SWNTs are excellent materials for biosensor development. Various SWNT-based biosensors have been developed that are sensitive to, for example, bacterial motifs, reactive oxygen species, metal ions, proteins, and neurotransmitters. The intentional introduction of sp³ defects by covalent functionalization has been shown to enhance the fluorescence properties of SWNTs and increase their PL quantum yield. At low densities, sp³ defects lead to new and bright emission bands (typically labeled as E₁₁*) that are red-shifted from the native excitonic E₁₁ emission. They provide additional fluorescence signals at different wavelengths and with different responses to changes in the nanotube environment and to analytes. Thus, they enable multimodal and ratiometric detection schemes. For the easily sorted and purified species of (6,5) SWNTs (diameter 0.76 nm), the E₁₁ emission occurs at ≈990 nm and the E₁₁ emission at ≈1140 nm in aqueous dispersion. Very recently, such sp³ defects were successfully used by Kim et al. as fluorescent probes to detect ovarian cancer and by Spreinat et al.