The spectroscopic properties of benzo[a]phenoxazin-5-one derivatives (3a-3m) including newly synthesized 3k-3m from 4-nitrosoaniline hydrochlorides and ethyl 1, 3-dihydroxy-2-naphthoate were studied. Compound 31 was converted into a covalent product with DNA, which had a blue shift of the fluorescence maximum. Compounds 3a-3k were found to undergo interaction with DNA and their complexes with DNA had a red shift of the fluorescence maximum and showed increasing melting temperature of DNA. Compound 3m-DNA had a blue shift of the fluorescence maximun to 3m and showed decreasing melting temperature of DNA.