The absorption and fluorescence spectra of 3-aminobenzo-1,2,4-triazine di-N-oxide (tirapazamine) have been recorded and exhibit a dependence on solvent that correlates with the Dimroth E_T30 parameter. Time-dependent density functional theory calculations reveal that the transition of tirapazamine in the visible region is π → π* in nature. The fluorescence lifetime is 98 ± 2 ps in water. The fluorescence quantum yield is ∼0.002 in water. The fluorescence of tirapazamine is efficiently quenched by electron donors via an electron-transfer process. Linear Stern–Volmer fluorescence quenching plots are observed with sodium azide, potassium thiocyanate, guanosine monophosphate and tryptophan (Trp) methyl ester hydrochloride. Guanosine monophosphate, tyrosine (Tyr) methyl ester hydrochloride and Trp methyl ester hydrochloride appear to quench the fluorescence at a rate greater than diffusion control implying that these substrates complex with tirapazamine in its ground state. This complexation was detected by absorption spectroscopy.