Herein, a strategy has been proposed to synthesis carbon dots (CDs) with blue fluorescence, and their phosphorescence is further activated in aqueous environment through CDs cooperating with cyanuric acid (CA). Significantly, the dual-emissive fluorescence and phosphorescence of CA-CDs could be effectively quenched by manganese dioxide (MnO 2 ) nanosheets through F o ¨ rster resonance energy transfer and dynamic quenching effect. Based on the generation of hydrogen peroxide by enzyme catalyzing the corresponding substrate, a universal sensing platform for detecting cholesterol and glucose has been originally established, which also broadened the avenue of assaying other targets. • Phosphorescence in aqueous was activated through CDs cooperating with CA. • A universal dual-channel sensing platform was originally established. • The sensing mechanism by CA-CDs was explored and clarified in detail. The current fluorescence assays are generally restricted by their background fluorescence and scattering light, thus room temperature phosphorescence (RTP) materials show the promising prospect towards biosensing. Herein, a facile strategy has been proposed to synthesize carbon dots (CDs) with blue fluorescence, which further achieved emitting phosphorescence in aqueous environment by cooperating with cyanuric acid (CA). Significantly, the dual-emissive fluorescence and phosphorescence of CA-CDs could be obviously quenched by manganese dioxide nanosheets through F o ¨ rster resonance energy transfer and dynamic quenching effect. On the basis of the generation of hydrogen peroxide by enzyme catalyzing the corresponding substrate, a universal sensing platform for detecting cholesterol and glucose was originally established. To be specific, the linear ranges were 0.02625∼2.1 mg mL −1 for cholesterol sensing, and 0.1∼20 mM for the detection of glucose, which exhibited general agreement with the physiological range, thus may broaden the avenue of assaying other targets.