A critical review of room temperature phosphorescence (RTP) is presented, with a focus on recent developments. RTP techniques including solid substrate-RTP (SS-RTP) and fluid-RTP (F-RTP), in which involved micelle-stabilized (MS-RTP), cyclodextrin induced-RTP (CD-RTP), sensitized and quenched-RTP (S/Q-RTP), colloid microcrystal-RTP (CM-RTP), microemulsion-RTP (ME-RTP), vesicles stabilized-RTP (VS-RTP) and non-protected fluid-RTP (NP-RTP) are discussed. These techniques have been widely used for organic trance analysis. In addition, the use of RTP as a powerful detection technique to develop new optical sensors and sensing systems for a wide variety of analytes and applications are reviewed. Next to the application of RTP in quantitative analysis is also examined. Finally, the application of RTP in nucleic acids detection and immunoassays, its use for the structural probing of protein conformations and for time-resolved microscopy of labeled biomolecules is proposed. The paper closes with a short evaluation of RTP and a discussion on some key areas of recent study and trends envisaged for future perspectives.