Tiagabine hydrochloride (TGB) is a clinically frequently used drug for anticonvulsion and reducing epileptic frequency. Over administration of TGB could bring about adverse effects, such as speech disorder, depression, and even suicidal tendencies. Therefore, accessible and sensitive assay for analysis of TGB becomes an urgent need toward guiding clinical medication. Here, we present the first report on fluorescence turn-on detection of TGB in urine testing. In this protocol, a fluorescent dye, perylene tetracarboxylic acid imide derivative (PTAI), is found specifically occupying the Sudlow site II of human serum albumin (HSA) and displays a new phenomenon of binding-induced quenching (BIQ). In presence of TGB, competitive binding of the TGB to the site II of HSA will trigger release of PTAI, thus successfully lighting up the fluorescence of PTAI. This label-free assay enjoys a broader working range (1–350 μM) and lower detection limit (0.218 μM) than the traditional liquid chromatography method and is uninterfered by the miscellaneous in the artificial urine. The BIQ probe highlights the merits of HSA as a quencher and a molecular recognition unit, and it opens up a way for studying drug-HSA interaction mechanism and noninvasive pharmaceutical testing.