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• A new type of phosphorescent material without metal or organic fluorophores. • Carbon-related defects leading the intersystem crossing-based phosphorescence. • The rigid silica network ensuring the longevity of the phosphorescent emission. • Time-gated afterglow imaging system eliminating the tissue-autofluorescence. • Clear visualization-based diagnosis of subcutaneous tumors by high-contrast imaging. Room-temperature phosphorescence (RTP) has tremendous potential in optics and photonics. Unlike fluorescence, RTP has substantial afterglow signals even after the excitation light is removed, which allows for extended acquisition times and higher signal-to-noise ratio under time-gated bioimaging. However, conventional RTP materials, both metal-containing and metal-free organic compounds, typically have limited photostability and inherent toxicity, making them unsuitable for long-term biological applications. Here, we report metal- and organic fluorophore-free silica nanoparticles (SNPs) that facilitate long-lived phosphorescence and exhibit RTP for high-contrast bioimaging. Polycondensation of silicon precursors and silyl biphenyls forms biphenyl-doped SNPs (bSNPs), and thermal decomposition of biphenyl moieties generates optically active defects in the biphenyl-bonded silicate network. The calcined bSNPs (C-bSNPs) have RTP-related biphenyl defects composed of carbon impurities, corresponding to spectroscopic measurements and ab initio calculations. Facile surface functionalization of defect-engineered C-bSNPs with tumor-targeting peptides while maintaining long-lived RTP allows for tissue autofluorescence-free in vivo bioimaging for cancer diagnosis, surpassing the limitations of continuous-wave imaging. [ABSTRACT FROM AUTHOR]