This study introduces sp³-functionalized (6,5) single-walled carbon nanotubes (SWNTs) as near-infrared luminescent probes for the detection and quantification of inorganic pyrophosphate (PPi). The SWNTs are functionalized with luminescent defects bearing an alkyne moiety, which exhibit a high sensitivity to Cu²⁺ ions. The presence of Cu²⁺ ions quenches the fluorescence via photoinduced electron transfer, while the addition of PPi reverses this quenching effect. The differences in the fluorescence response of the sp³-defect emission compared to the pristine nanotube emission enable reproducible ratiometric measurements over a wide concentration range. Biocompatible, phospholipid-polyethylene glycol-coated SWNTs are used to detect PPi in cell lysate and monitor DNA synthesis in a polymerase chain reaction (PCR). The robust ratiometric and near-infrared luminescent probe for PPi shows potential for the design of nanotube-based biosensors. The study also explores the mechanism of PL quenching and recovery, suggesting that fast photoinduced electron transfer is the primary cause. The developed sensor remains fully operational with a detection window over two orders of magnitude when made biocompatible, making it suitable for various applications, including PCR quality control and in vivo detection of PPi.This study introduces sp³-functionalized (6,5) single-walled carbon nanotubes (SWNTs) as near-infrared luminescent probes for the detection and quantification of inorganic pyrophosphate (PPi). The SWNTs are functionalized with luminescent defects bearing an alkyne moiety, which exhibit a high sensitivity to Cu²⁺ ions. The presence of Cu²⁺ ions quenches the fluorescence via photoinduced electron transfer, while the addition of PPi reverses this quenching effect. The differences in the fluorescence response of the sp³-defect emission compared to the pristine nanotube emission enable reproducible ratiometric measurements over a wide concentration range. Biocompatible, phospholipid-polyethylene glycol-coated SWNTs are used to detect PPi in cell lysate and monitor DNA synthesis in a polymerase chain reaction (PCR). The robust ratiometric and near-infrared luminescent probe for PPi shows potential for the design of nanotube-based biosensors. The study also explores the mechanism of PL quenching and recovery, suggesting that fast photoinduced electron transfer is the primary cause. The developed sensor remains fully operational with a detection window over two orders of magnitude when made biocompatible, making it suitable for various applications, including PCR quality control and in vivo detection of PPi.