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.