Matrix-free room temperature phosphorescence (RTP) materials have generated considerable interest in sensing, anti-counterfeiting and bioimaging due to long lifetime and large Stokes shift. However, spin-forbidden transition and non-radiative transition make RTP challenging to implement. In this work, matrix-free RTP carbon dots (CDs) co-doped with boron and nitrogen were prepared through the microwave-assisted strategy. The CDs realize RTP without any protective matrix. This may be attributed to the stable and rigid structure formed during the heating polymerization process, which can protect the triplet excited state from being quenched by oxygen and water in the air environment. The fluorescence and phosphorescence emission wavelength of the as-prepared CDs were observed at 532 nm and 596 nm with the excitation wavelength of 476 nm and 514 nm, respectively, showing a long afterglow lifetime of 735.3 ms. Nitrogen doping has a crucial effect on the luminescence of RTP, and the increase of nitrogen content facilitates the n-π* transition and improve the efficiency of intersystem crossing (ISC). Importantly, the ratiometric fluorescence method for determining riboflavin has been established based on the Förster resonance energy transfer (FRET) between the CDs (Donor) and riboflavin (Acceptor), with a linear relationship in the range of 9.52–85.71 μM and the limit of detection (LOD) of 19.9 nM. Additionally, the phosphorescent property of the CDs was exploited to demonstrate the potential applications in the field of information encryption.