Currently, the synthesis and applications of room-temperature phosphorescent (RTP) materials are promising, but challenging. Herein, a kind of ultra-long phosphorescent carbon dots (P-CDs) were rapidly prepared through a facile microwave-directed method. In particular, the yellow-green phosphorescence of the P-CDs was observed for up to 9 s, and their fluorescence and phosphorescence were observed at 418 nm and 525 nm, respectively, using the same excitation at 354 nm. Moreover, the RTP intensity of the P-CDs gradually decreased with the increasing pH because of the protonation-dissociated hydrogen bonds and disturbed phosphorescence sources, whereas their fluorescence exhibited general stability over a wide pH range of 2.1–12.68. Significantly, we found that both the fluorescence and the phosphorescence of the P-CDs were quenched by the introduction of tetracyclines (TCs). Specifically, the P-CDs described herein have abundant –COOH and –OH groups on their surfaces, which facilitate the linkage of CDs with TCs, thus resulting in the occurrence of an inner filter effect process (IFE). Moreover, the P-CDs showed potential as an ideal candidate for building up dual-channel assays towards the targets.