The fluorescent lapachones were obtained from lapachol, a natural product extracted from the heartwood of Tabebuia sp. (Tecoma). The extracted lapachol was oxidized and cyclized to obtain the compounds β-lapachone and nor-β-lapachone, and treatment with terephthalaldehyde led to the final products with ∼ 60% yield. The photophysical studies showed absorption maxima in the violet region (∼400 nm) and fluorescence emission maxima depending on the solvent, ranging from 430 to 575 nm with a relatively large positive solvatochromism, confirmed by the Lippert-Mataga plot. Theoretical calculations corroborate the experimental observations regarding the ICT mechanism. The imidazoles showed a selective response to bisulfite ion (HSO3−) over other common anions, displaying a large change in both fluorescence and absorption spectra immediately after the addition of bisulfite, proving their potential application in tracing the bisulfite in future biological systems. The binding capacity of the imidazoles with human or bovine serum albumin (HSA and BSA, respectively) is spontaneous, weak (102–103 M−1 range), and subdomains IIA (Site I) and IB (Site III) are the main binding regions for the imidazoles depending on the nature of the metabolite or drug previously complexed with the albumin structure. Overall, both albumins have the same capacity to interact with the imidazoles under study.