We studied the spectral properties of 4′-6-diamidino-2-phenylindole (DAPI) in poly (vinyl alcohol) (PVA) films. Absorption and fluorescence spectra, emission and excitation spectra, quantum yield, and fluorescence lifetime have been characterized. An efficient room temperature phosphorescence (RTP) of DAPI has been observed with UV and blue light excitations. A few hundred millisecond phosphorescence lifetime enables a gated detection with sufficient background reduction. We found the phosphorescent Quantum Yield of DAPI in PVA Film to be 0.0009.