Hypoxia is a parameter related to many diseases. Ratiometric hypoxia probes often rely on a combination of an O2‐insensitive fluorophore and an O2‐sensitive phosphor in a polymer matrix, which require high cost and multi‐step synthesis of transition metal complexes. The two‐chromophore hypoxia probes encounter unfavorable energy transfer processes and different stabilities of the chromophores. Reported herein is a pure organic ratiometric hypoxia nanoprobe, assembled by a monochromophore, naphthalimide ureidopyrimidinone (BrNpA‐UPy), bridged by a bis‐UPy‐functionalized benzyl skeleton. The joint factors of quadruple hydrogen bonding, the rigid backbone of UPy, and bromine substitution of the naphthalimide derivative facilitate bright phosphorescence (ΦP=7.7 %, τP=3.2 ms) and fluorescence of the resultant nanoparticles (SNPs) at room temperature, which enable accurate, ratiometric, sensitive oxygen detection (Ksv=189.6 kPa−1) in aqueous solution as well as in living HeLa cells. An organic hypoxia nanoprobe assembled by quadruple hydrogen bonds, shows efficient long‐lived phosphorescence (ΦP=7.7 %, τP=3.2 ms) and fluorescence from a monochromophore at room temperature and can be used for oxygen detection in water and living cells. This is the first example of ratiometric hypoxia sensing by supramolecular assemblies of an organic monochromophore.