The first example of a ratiometric optical oxygen nanoprobe based on a hydrogen-bonded supramolecular polymer has been reported. The supramolecular polymer based nanoprobe (SPNP) is prepared from the co-assembly of a bis-ureidopyrimidinone (bis-UPy)-containing phosphorescent indicator (Por(Pd)-bisUPy), fluorescent reference dye (BF2-bisUPy), and skeleton unit (DPA-bisUPy) through quadruple hydrogen bonds by a mini-emulsion method. The water-dispersible SPNP is highly sensitive to oxygen (Q = 95%), with full reversibility, excellent storage stability and photostability, and good cell-penetrating ability, and exhibits low cytotoxicity toward living cells. The preparation of the SPNP is straightforward and its function is easily tuned by changing the monomeric structure. This work is expected to lead to the design of other SPNPs for other important analytes in biological systems.